Identification of Pathogenic Copy Number Variants in Mexican Patients With Inherited Retinal Dystrophies Applying an Exome Sequencing Data-Based Read-Depth Approach

Abstract

Background: Retinal dystrophies (RDs) are the most common cause of inherited blindness worldwide and are caused by genetic defects in about 300 different genes. While targeted next-generation sequencing (NGS) has been demonstrated to be a reliable and efficient method to identify RD disease-causing variants, it doesn't routinely identify pathogenic structural variant as copy number variations (CNVs). Targeted NGS-based CNV detection has become a crucial step for RDs molecular diagnosis, particularly in cases without identified causative single nucleotide or Indels variants. Herein, we report the exome sequencing (ES) data-based read-depth bioinformatic analysis in a group of 30 unrelated Mexican RD patients with a negative or inconclusive genetic result after ES.

Methods: CNV detection was performed using ExomeDepth software, an R package designed to detect CNVs using exome data. Bioinformatic validation of identified CNVs was conducted through a commercially available CNV caller. All identified candidate pathogenic CNVs were orthogonally verified through quantitative PCR assays.

Results: Pathogenic or likely pathogenic CNVs were identified in 6 out of 30 cases (20%), and of them, a definitive molecular diagnosis was reached in 5 cases, for a final diagnostic rate of ~17%. CNV-carrying genes included CLN3 (2 cases), ABCA4 (novel deletion), EYS, and RPGRIP1.

Conclusions: Our results indicate that bioinformatic analysis of ES data is a reliable method for pathogenic CNV detection and that it should be incorporated in cases with a negative or inconclusive molecular result after ES.

Keywords: copy number variation; deletion; exome sequencing; retinal dystrophy.

FIGURE 1

CLN3 intragenic deletion analysis. (A) Heterozygous deletion of exons 8 and 9 of the CLN3 gene identified by ExomeDepth in the ES data from patient 1 (Table 1). The red crosses show the ratio of observed/expected number of reads for the test sample. The gray‐shaded region shows the estimated 99% confidence interval for this observed ratio in the absence of CNV call. (B) Bioinformatic confirmation of the CLN3 deletion through Rainbow tool. The median depth of the sample (blue line, arrow) is compared with a set (at least 30) of reference samples. In this case, the predicted copy number is 1, indicating a heterozygous deletion of CLN3 exons 8 and 9. (C) Quantitative PCR of DNA concentration ratio between CLN3 and a reference gene (PIKFYVE) confirming the presence of the deletion in exon 8 (exon 9 deletion is not shown). (D) qPCR control assay in DNA from patient 1 demonstrating normal gene dosage for CLN3 exon 10 as compared with the reference gene (PIKFYVE).

FIGURE 2

RPGRIP1 intragenic deletion analysis. (A) Heterozygous deletion of exons 3–18 of the RPGRIP1 gene identified by ExomeDepth in the ES data from patient 3 (Table 1). The red crosses show the ratio of observed/expected number of reads for the test sample. The gray‐shaded region shows the estimated 99% confidence interval for this observed ratio in the absence of CNV call. (B) Bioinformatic confirmation of the RPGRIP1 deletion through Rainbow tool. The median depth of the sample (blue line, arrow) is compared with a set (at least 30) of reference samples. In this case, the predicted copy number is 1, indicating a heterozygous deletion of RPGRIP1 exons 3–18. (C) Quantitative PCR of DNA concentration ratio between RPGRIP1 and a reference gene (PIKFYVE) confirming an exon 15 deletion (qPCRs of the other deleted exons are not shown). (D) qPCR control assay in DNA from patient 3 demonstrating normal gene dosage for RPGRIP1 exon 19 as compared with the reference gene (PIKFYVE).

FIGURE 3

Intragenic ABCA4 deletion analysis. (A) Heterozygous deletion of exons 15–23 of the ABCA4 gene identified by ExomeDepth in the ES data from patient 4 (Table 1). The red crosses show the ratio of observed/expected number of reads for the test sample. The gray‐shaded region shows the estimated 99% confidence interval for this observed ratio in the absence of CNV call. (B) Bioinformatic confirmation of the ABCA4 deletion through Rainbow tool. The median depth of the sample (blue line, arrow) is compared with a set (at least 30) of reference samples. In this case, the predicted copy number is 1, indicating a heterozygous deletion of ABCA4 exons 15–23. (C) Quantitative PCR of DNA concentration ratio between ABCA4 and a reference gene (PIKFYVE) confirming the presence of the deletion in exon 18 (qPCRs of the other deleted exons are not shown. (D) qPCR control assay in DNA from patient 4 demonstrating normal gene dosage for ABCA4 exon 24 as compared with the reference gene (PIKFYVE).

FIGURE 4

Intragenic EYS deletion analysis. (A) Heterozygous deletion of exon 24 of the EYS gene identified by ExomeDepth in the ES data from patient #5 (Table 1). The red crosses show the ratio of observed/expected number of reads for the test sample. The gray‐shaded region shows the estimated 99% confidence interval for this observed ratio in the absence of CNV call. (B) Bioinformatic confirmation of the EYS deletion through Rainbow tool. The median depth of the sample (blue line, arrow) is compared with a set (at least 30) of reference samples. In this case, the predicted copy number is 1, indicating a heterozygous deletion of EYS exon 24. (C) Quantitative PCR of DNA concentration ratio between EYS and a reference gene (PIKFYVE) confirming the presence of the deletion in exon 24 in DNA from patient #5.