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

<div><p>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 <i>EYS</i> 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 <i>RP1</i>, 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.</p></div

Mutational spectrum in RP and LCA patients.

<p>Percentages refer to patients with mutations in the respective gene that are considered causative. The distribution of causative mutations across many genes, each contributing a relatively small fraction to the mutational spectrum, confirms the extensive genetic heterogeneity of retinal dystrophies. Note that the three patients that were found to carry X-linked mutations are not contained in the schemes A – B. <b>A.</b> arRP. <b>B.</b> adRP. Note that the percentages refer to a relatively small adRP cohort in this study. <b>C.</b> LCA. <b>D.</b> Functional categorization of genes that were found to carry causative mutations in our study. Mutations in genes encoding components of the photoreceptor’s connecting cilium and associated structures were predominant.</p

Comparison of this study with previous NGS studies on retinal dystrophies.

*<p>Positive controls not included.</p>**<p>Additional samples from the same families not included. Gene numbers in brackets include additionally screened candidate genes that are not yet proven retinal disease genes. BBS, Bardet-Biedl syndrome; CRD, cone-rod dystrophy; CD, cone dystrophy; CSNB, congenital stationary night blindness; FA, fundus albipunctatus; STGD, Morbus Stargardt; USH, Usher syndrome.</p

Causative mutations and putatively pathogenic variants identified in this study.

<p>Causative alleles are being listed as “allele 1” and “allele 2” in resolved cases. Additional alleles are shown if the minor allele frequency is below 3% and if <i>in silico</i> prediction suggests putative pathogenicity. The inheritance pattern was largely delineated from pedigree informations. In patients 22, 23, 77, 100, 116 and 119, the true mode of inheritance had not been evident from the pedigree information and was finally deduced from the genotype. a, this study. References for studies cited in this table can be found in the Supplementary Material (References S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone.0078496.s001" target="_blank">File S1</a>). n.d., not defined; f, female; m, male; ar, autosomal recessive; ad, autosomal dominant; s, sporadic. Xl, X-linked. Cau, Caucasian; Ger, Germany; Tur, Turkey; KSA, Kingdom of Saudi Arabia; Pol, Poland; Au, Austria; Syr, Syria; Pak, Pakistan; DRC, Democratic Republic of the Congo; Mor, Morocco; UAE, United Arab Emirates; E-Eur, East Europe; SE-Eur, Southeast Europe.</p

Hemizygosity of a <i>CRX</i> mutation in a recessive consanguineous LCA family.

<p><b>A.</b> Compound-heterozygosity for a potentially protein-extending no-stop mutation (c.899A>G/p.(*300Trpext*118); here designated as Ext) abrogating the natural termination codon in exon 4 and a deletion of the same exon (delE4) <i>in trans</i> in patient 110 and her brother. <b>B.</b> Graphical view of the LOD score calculation from genomewide SNP mapping for this family previous to NGS testing: Genomewide homozygosity mapping prior to NGS did not identify a clear candidate locus. The combined maximum parametric LOD score of 2.4 was not obtained. <b>C.</b> Scheme of the <i>CRX</i> gene and coverage plots for CNV analysis from NGS data (Illumina MiSeq), indicating a heterozygous deletion of exon 4 (upper panel, absolute coverage based on read count; lower panel, SeqNext CNV analysis). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone-0078496-g002" target="_blank">Figure 2C</a>. <b>D.</b> Schematic representation of the mapped sequencing reads for the no-stop mutation (Integrative Genomics Viewer). The mutation (arrow) was present in all 65 reads covering this region of the gene and therefore appeared homozygous. <b>E.</b> Electropherograms from Sanger sequencing of the no-stop mutation with hemizygosity in patient 110 (upper panel) and heterozygosity in her mother (lower panel). <b>F.</b> Summary of the disease-causing genetic constellation in patient 110 and her brother (superimposition on parental alleles).</p

Different arRP scenarios implicating truncating <i>RP1</i> mutations with diverse impact on disease.

<p><b>A.</b> Pedigree of patient 25 whose arRP is caused by two truncating recessive <i>RP1</i> alleles. In addition, the patient carries a heterozygous <i>CDH23</i> nonsense mutation that has been reported in USH1 patients but is probably unrelated to disease here. <b>B.</b> LCA in patient 124 is due to homozygosity for the founder mutation p.Gln301* in <i>TULP1</i>. Heterozygosity for the <i>RP1</i> nonsense mutation p.Glu1750* likely reflects accidental carriership. It likely represents a recessive loss-of-function allele. Dotted horizontal line: likely consanguinity. <b>C.</b> Compound heterozygosity for two truncating <i>PROM1</i> mutations can be considered pathogenic in arRP patient 55. The <i>RP1</i> nonsense mutation p.Gln2102* locates near the C-terminus and likely represents an NMD-insensitive non-pathogenic variant. <b>D.</b> Scheme of the RP1 protein and overview of truncating <i>RP1</i> mutations reported in this study (mutations shown in A – C in red). The four classes of <i>RP1</i> truncating mutations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078496#pone.0078496-Chen1" target="_blank">[31]</a> are displayed. Class I, NMD-sensitive truncations; class II, NMD-insensitive truncating mutations representing the majority of pathogenic truncation mutations in <i>RP1</i> (dominant negative pathomechanism); class III, NMD-insensitive truncation mutations representing loss-of-function arRP mutations; class IV, NMD-insensitive, non-pathogenic truncations located 3′ of p.1816. CP, “critical position”: 65-residue region between p.1751 and p.1816 containing a yet undefined protein residue before which truncation causes disease.</p