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

TY - JOUR

T1 - 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

AU - Eisenberger, Tobias

AU - Neuhaus, Christine

AU - Khan, Arif O

AU - Decker, Christian

AU - Preising, Markus N

AU - Friedburg, Christoph

AU - Bieg, Anika

AU - Gliem, Martin

AU - Charbel Issa, Peter

AU - Holz, Frank G

AU - Baig, Shahid M

AU - Hellenbroich, Yorck

AU - Galvez, Alberto

AU - Platzer, Konrad

AU - Wollnik, Bernd

AU - Laddach, Nadja

AU - Ghaffari, Saeed Reza

AU - Rafati, Maryam

AU - Botzenhart, Elke

AU - Tinschert, Sigrid

AU - Börger, Doris

AU - Bohring, Axel

AU - Schreml, Julia

AU - Körtge-Jung, Stefani

AU - Schell-Apacik, Chayim

AU - Bakur, Khadijah

AU - Al-Aama, Jumana Y

AU - Neuhann, Teresa

AU - Herkenrath, Peter

AU - Nürnberg, Gudrun

AU - Nürnberg, Peter

AU - Davis, John S

AU - Gal, Andreas

AU - Bergmann, Carsten

AU - Lorenz, Birgit

AU - Bolz, Hanno J

PY - 2013/1/1

Y1 - 2013/1/1

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pone.0078496

DO - 10.1371/journal.pone.0078496

M3 - SCORING: Journal article

C2 - 24265693

VL - 8

SP - e78496

JO - PLOS ONE

JF - PLOS ONE

SN - 1932-6203

IS - 11

ER -