Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders

Abstract

Simultaneous analysis of multiple genes using next-generation sequencing (NGS) technology has become widely available. Copy-number variations (CNVs) in disease-associated genes have emerged as a cause for several hereditary disorders. CNVs are, however, not routinely detected using NGS analysis. The aim of this study was to assess the diagnostic yield and the prevalence of CNVs using our panel of Hereditary Thoracic Aortic Disease (H-TAD)-associated genes. Eight hundred ten patients suspected of H-TAD were analyzed by targeted NGS analysis of 21 H-TAD associated genes. In addition, the eXome hidden Markov model (XHMM; an algorithm to identify CNVs in targeted NGS data) was used to detect CNVs in these genes. A pathogenic or likely pathogenic variant was found in 66 of 810 patients (8.1%). Of these 66 pathogenic or likely pathogenic variants, six (9.1%) were CNVs not detectable by routine NGS analysis. These CNVs were four intragenic (multi-)exon deletions in MYLK, TGFB2, SMAD3, and PRKG1, respectively. In addition, a large duplication including NOTCH1 and a large deletion encompassing SCARF2 were detected. As confirmed by additional analyses, both CNVs indicated larger chromosomal abnormalities, which could explain the phenotype in both patients. Given the clinical relevance of the identification of a genetic cause, CNV analysis using a method such as XHMM should be incorporated into the clinical diagnostic care for H-TAD patients.

Keywords: copy-number variations; eXome hidden Markov model; genetics; thoracic aortic aneurysm; thoracic aortic dissection.

Figure 1

Genomic copy‐number variants in H‐TAD patients based on XHMM analysis. PCA: principal‐component analysis; XHMM: eXome hidden Markov model. A, MYLK gene; deletion of exons 17 and 18. B, PRKG1 gene; deletion of exon 3. C, SMAD3; deletion of exon 6. D, TGFB2; deletion of exons 4, 5, 6, and 7. E, NOTCH1 gene; whole gene duplication. F, SCARF2 gene; whole gene deletion. Graphic representation of the copy‐number variants in each gene based on XHMM analysis. Horizontal axis indicates physical position of the CNVs. Vertical axis indicates sample Z‐score of PCA‐normalized read depth. Deletions are colored in red, and duplications are colored in green

Figure 1

Genomic copy‐number variants in H‐TAD patients based on XHMM analysis. PCA: principal‐component analysis; XHMM: eXome hidden Markov model. A, MYLK gene; deletion of exons 17 and 18. B, PRKG1 gene; deletion of exon 3. C, SMAD3; deletion of exon 6. D, TGFB2; deletion of exons 4, 5, 6, and 7. E, NOTCH1 gene; whole gene duplication. F, SCARF2 gene; whole gene deletion. Graphic representation of the copy‐number variants in each gene based on XHMM analysis. Horizontal axis indicates physical position of the CNVs. Vertical axis indicates sample Z‐score of PCA‐normalized read depth. Deletions are colored in red, and duplications are colored in green

Figure 1

Genomic copy‐number variants in H‐TAD patients based on XHMM analysis. PCA: principal‐component analysis; XHMM: eXome hidden Markov model. A, MYLK gene; deletion of exons 17 and 18. B, PRKG1 gene; deletion of exon 3. C, SMAD3; deletion of exon 6. D, TGFB2; deletion of exons 4, 5, 6, and 7. E, NOTCH1 gene; whole gene duplication. F, SCARF2 gene; whole gene deletion. Graphic representation of the copy‐number variants in each gene based on XHMM analysis. Horizontal axis indicates physical position of the CNVs. Vertical axis indicates sample Z‐score of PCA‐normalized read depth. Deletions are colored in red, and duplications are colored in green

Figure 2

Further characterization of XHMM results by additional (cyto‐) genetic testing. BAF, B allele frequency; Chr, chromosome; der, derivate chromosome; LLR, log R ratio; FISH, fluorescence in situ hybridization. A, SNP array profile of chromosomes 7 and 9 are shown on the left. The top plot of each image shows the LRR, which provides an estimation of the copy number for each marker aligned to its chromosomal position. The bottom plot of each image shows the BAF for each SNP aligned to its chromosomal position. SNP array analysis revealed a terminal copy‐number loss at 7p22.3 (2Mb; GRCh37; chr7:43360‐2067625) indicated with a red arrow and a terminal copy‐number gain at 9q33.3–q34.43 (11.8Mb; GRCh37; chr9:129172353–141020389) indicated with a green arrow. Chromosomes 7 and 9 from the index (left) with the unbalanced translocation and the father (right) carrying the balanced translocation are shown on the right. The breakpoints of the reciprocal translocation are indicated with an arrow. The index has the derivative chromosome 7 lacking a short segment from the short arm of chromosome 7 that is replaced by an extra copy of a terminal segment of chromosome 9q. The father has two derivative chromosomes 7 and 9, each carrying a segment of the other chromosome. B, SNP array profile of chromosome 22 is shown on the left. SNP array analysis revealed a copy‐number loss at 22q11.2 (3.2Mb; GRCh37; chr22:20779645_20792061) indicated with a red arrow. The results of metaphase FISH on blood from the mother is presented on the right. The 22q11.2 region is recognized by the HIRA probe, producing a red signal. The green signal is from the ARSA probe hybridizing with the ARSA gene on chromosome band 22q13.33. The 22q11.2 deletion is indicated by a blue arrow. Metaphase FISH analysis revealed that the mother is also a carrier of the 22q11.2 deletion (ish del(22)(q11.2q11.2)(HIRA‐))