Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease

Abstract

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Keywords: CNV; GATK-gCNV; copy number variant; diagnostic yield; exome; variant classification.

Figure 1

Exome copy number plot and reads visualization for examples of causal copy number variants (CNVs) in the Broad CMG cohort (A) Individual affected with retinitis pigmentosa with a homozygous single-exon deletion in CRB1 (chr1:197,438,450–197,439,442x0, quality score [QS] = 120) identified on exome. To evaluate the quality of the CNV, the patient’s copy number (CN) level (in red) was compared to a cluster of other samples with similar read depth that act as controls. The proband’s CN is decreased compared to the background cluster, compatible with a homozygous deletion. Y axis: CN. (B) As breakpoints are located within the exome data, manual inspection of read data from the individual from (A) using the Integrated Genomics Viewer (IGV) showed discordant read pairs, split reads, and complete absence of coverage, compatible with a homozygous exon 10 deletion also including part of upstream exon 9 in CRB1 (chr1:197,435,257–197,441,674x0 [GenBank: NM_201253.3]). Cov, coverage. (C) Individual with multiple congenital anomalies and a heterozygous deletion of four exons in RAB3GAP1 (Warburg micro syndrome) (red, chr2:135,162,318–135,164,794x1, QS = 92) in trans with a frameshift variant in RAB3GAP1 (not shown, c.2393_2394del [GenBank: NM_012233.3] [p.Leu798ArgfsTer7]), both identified by exome. The presence of the deletion was validated by droplet digital PCR. Y axis: CN. (D) Individual with a neurodevelopmental disorder with a de novo 2.6 Mb heterozygous 1q43q44 deletion (red, chr1:242,523,991–245,156,781x1, QS = 3077) identified on exome. The presence of this deletion was validated by quantitative PCR. Y axis: CN. (E) Individual with a neurodevelopmental disorder with a de novo 2.1 Mb 22q11.2 duplication (red, chr22:18,985,739–21,081,116x3, QS = 3077) identified on exome. The presence of this duplication was validated by chromosomal microarray (CMA). Y axis: CN. All coordinates on GRCh38.

Figure 2

Flowchart illustrating how points were scored for copy number variants (CNVs) that followed an X-linked inheritance pattern We incorporated sex of proband, parental genotype, and parental affected status to score both the proband in which the X-linked variant was identified and, if applicable, any individual in the published literature or public databases that had variants of similar genomic content to the variant of interest. The points for each case could be increased or decreased based on phenotype specificity, up to 0.45 points.

Figure 3

Characteristics of copy number variants (CNVs) across the entire callset and the subset of causal CNVs (A) Number of high-confidence CNVs by estimated size that were identified in the Broad CMG exome callset of 6,633 families sequenced between 2016 and 2021. Large CNVs tend to be fragmented into multiple small GATK-gCNV calls, accounting for why there are no CNVs in the >10 Mb category of the graph. These CNVs were interpreted as being part of the same underlying event when looking at the copy number plot and/or validation methods and are presented that way in Figures 3B and 3C. DEL, deletion; DUP, duplication. (B) Mode of inheritance and number of genes involved in each CNV in 171 families in which the CNV was interpreted as causal. The number of genes included in each interval was chosen based on cutoffs suggested for CNV scoring in section 3 of the Riggs et al. ACMG/ClinGen standards.AD, autosomal dominant; AR, autosomal recessive; XL, X-linked. (C) CNV classification by estimated size in 171 families in which the CNV was interpreted as causal by the multidisciplinary team. The causal CNVs consisted of 140 deletions, 15 duplications, 3 insertions (miscalled as deletion by GATK-gCNV), and 13 complex structural variants (SVs). We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as variants of uncertain significance (VUSs).