title: Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.
abstract: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.
authors.affiliation: Bioscientia Center for Human Genetics, Ingelheim, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Eisenberger
authors.forename: Christine
authors.initials: C
authors.lastname: Neuhaus
authors.forename: Arif O
authors.initials: AO
authors.lastname: Khan
authors.forename: Christian
authors.initials: C
authors.lastname: Decker
authors.forename: Markus N
authors.initials: MN
authors.lastname: Preising
authors.forename: Christoph
authors.initials: C
authors.lastname: Friedburg
authors.forename: Anika
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authors.lastname: Bieg
authors.forename: Martin
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authors.lastname: Gliem
authors.forename: Peter
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authors.lastname: Charbel Issa
authors.forename: Frank G
authors.initials: FG
authors.lastname: Holz
authors.forename: Shahid M
authors.initials: SM
authors.lastname: Baig
authors.forename: Yorck
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authors.lastname: Hellenbroich
authors.forename: Alberto
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authors.lastname: Galvez
authors.forename: Konrad
authors.initials: K
authors.lastname: Platzer
authors.forename: Bernd
authors.initials: B
authors.lastname: Wollnik
authors.forename: Nadja
authors.initials: N
authors.lastname: Laddach
authors.forename: Saeed Reza
authors.initials: SR
authors.lastname: Ghaffari
authors.forename: Maryam
authors.initials: M
authors.lastname: Rafati
authors.forename: Elke
authors.initials: E
authors.lastname: Botzenhart
authors.forename: Sigrid
authors.initials: S
authors.lastname: Tinschert
authors.forename: Doris
authors.initials: D
authors.lastname: Börger
authors.forename: Axel
authors.initials: A
authors.lastname: Bohring
authors.forename: Julia
authors.initials: J
authors.lastname: Schreml
authors.forename: Stefani
authors.initials: S
authors.lastname: Körtge-Jung
authors.forename: Chayim
authors.initials: C
authors.lastname: Schell-Apacik
authors.forename: Khadijah
authors.initials: K
authors.lastname: Bakur
authors.forename: Jumana Y
authors.initials: JY
authors.lastname: Al-Aama
authors.forename: Teresa
authors.initials: T
authors.lastname: Neuhann
authors.forename: Peter
authors.initials: P
authors.lastname: Herkenrath
authors.forename: Gudrun
authors.initials: G
authors.lastname: Nürnberg
authors.forename: Peter
authors.initials: P
authors.lastname: Nürnberg
authors.forename: John S
authors.initials: JS
authors.lastname: Davis
authors.forename: Andreas
authors.initials: A
authors.lastname: Gal
authors.forename: Carsten
authors.initials: C
authors.lastname: Bergmann
authors.forename: Birgit
authors.initials: B
authors.lastname: Lorenz
authors.forename: Hanno J
authors.initials: HJ
authors.lastname: Bolz
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D056915:DNA Copy Number Variations; D005091:Exons / Q000235:genetics*; D005260:Female; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D058499:Retinal Dystrophies / Q000175:diagnosis / Q000235:genetics*; D017422:Sequence Analysis, DNA; D055815:Young Adult