Clinical Impact of Copy Number Variation on the Genetic Diagnosis of Syndromic Aortopathies.

Abstract

HomeCirculation: Genomic and Precision MedicineVol. 14, No. 4Clinical Impact of Copy Number Variation on the Genetic Diagnosis of Syndromic Aortopathies Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBClinical Impact of Copy Number Variation on the Genetic Diagnosis of Syndromic Aortopathies Norifumi Takeda, MD, PhD Ryo Inuzuka, MD, PhD Hiroki Yagi, MD, PhD Hiroyuki Morita, MD, PhD Masahiko Ando, MD, PhD Haruo Yamauchi, MD, PhD Yuki Taniguchi, MD, PhD Kristine Joyce Porto, Tsubasa Kanaya, MD Hiroyuki Ishiura, MD, PhD Jun Mitsui, MD, PhD Shoji Tsuji, MD, PhD Tatsushi Toda, MD, PhD Minoru Ono, MD, PhD Issei KomuroMD, PhD Norifumi TakedaNorifumi Takeda Norifumi Takeda, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Email E-mail Address: [email protected] Department of Cardiovascular Medicine (N.T., H. Yagi, H.M., T.K., I.K.), University of Tokyo Hospital, Japan. *N. Takeda and R. Inuzuka contributed equally. Search for more papers by this author , Ryo InuzukaRyo Inuzuka https://orcid.org/0000-0001-7501-689X Department of Pediatrics (R.I.), University of Tokyo Hospital, Japan. *N. Takeda and R. Inuzuka contributed equally. Search for more papers by this author , Hiroki YagiHiroki Yagi https://orcid.org/0000-0001-6615-6196 Department of Cardiovascular Medicine (N.T., H. Yagi, H.M., T.K., I.K.), University of Tokyo Hospital, Japan. Search for more papers by this author , Hiroyuki MoritaHiroyuki Morita https://orcid.org/0000-0003-0879-5576 Department of Cardiovascular Medicine (N.T., H. Yagi, H.M., T.K., I.K.), University of Tokyo Hospital, Japan. Search for more papers by this author , Masahiko AndoMasahiko Ando https://orcid.org/0000-0002-5481-3782 Department of Cardiac Surgery (M.A., H. Yamauchi, M.O.), University of Tokyo Hospital, Japan. Search for more papers by this author , Haruo YamauchiHaruo Yamauchi Department of Cardiac Surgery (M.A., H. Yamauchi, M.O.), University of Tokyo Hospital, Japan. Search for more papers by this author , Yuki TaniguchiYuki Taniguchi https://orcid.org/0000-0002-2329-123X Department of Orthopedic Surgery (Y.T.), University of Tokyo Hospital, Japan. Search for more papers by this author , Kristine Joyce PortoKristine Joyce Porto https://orcid.org/0000-0001-8262-0403 Department of Neurology (K.J.P., H.I., J.M., S.T., T.T.), University of Tokyo Hospital, Japan. Search for more papers by this author , Tsubasa KanayaTsubasa Kanaya https://orcid.org/0000-0001-8957-9239 Department of Cardiovascular Medicine (N.T., H. Yagi, H.M., T.K., I.K.), University of Tokyo Hospital, Japan. Search for more papers by this author , Hiroyuki IshiuraHiroyuki Ishiura https://orcid.org/0000-0003-2975-7309 Department of Neurology (K.J.P., H.I., J.M., S.T., T.T.), University of Tokyo Hospital, Japan. Search for more papers by this author , Jun MitsuiJun Mitsui https://orcid.org/0000-0001-7425-4765 Department of Neurology (K.J.P., H.I., J.M., S.T., T.T.), University of Tokyo Hospital, Japan. Search for more papers by this author , Shoji TsujiShoji Tsuji Department of Neurology (K.J.P., H.I., J.M., S.T., T.T.), University of Tokyo Hospital, Japan. Search for more papers by this author , Tatsushi TodaTatsushi Toda Department of Neurology (K.J.P., H.I., J.M., S.T., T.T.), University of Tokyo Hospital, Japan. Search for more papers by this author , Minoru OnoMinoru Ono Department of Cardiac Surgery (M.A., H. Yamauchi, M.O.), University of Tokyo Hospital, Japan. Search for more papers by this author , and Issei KomuroIssei Komuro Correspondence to: Issei Komuro, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Email E-mail Address: [email protected] https://orcid.org/0000-0002-0714-7182 Department of Cardiovascular Medicine (N.T., H. Yagi, H.M., T.K., I.K.), University of Tokyo Hospital, Japan. Search for more papers by this author Originally published30 Jul 2021https://doi.org/10.1161/CIRCGEN.121.003458Circulation: Genomic and Precision Medicine. 2021;14:e003458Next-generation sequencing technology provided a significant step forward in precision medicine of syndromic aortopathies. Up to 90% of patients with a clinical diagnosis of Marfan syndrome have pathogenic variants in the FBN1 gene and 6 TGF-β (transforming growth factor β) signal-related genes—TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, and SMAD2—have been reported to cause Loeys–Dietz syndrome.1 However, the genetic basis for the remaining 5% to 10% of syndromic aortopathies remains to be elucidated, and thus further investigation is important to identify the causative genes as well as the mechanisms responsible for the development. In this study, we aimed to reconfirm the significance of copy number variation (CNV) in syndromic aortopathies,2 because an apparent small impact of CNV on diagnostic yield may arise from insufficient power of the existing next-generation sequencing-based CNV detection tools.Next-generation sequencing-based genetic testing was performed in 84 unrelated patients diagnosed or suspected of syndromic aortopathies, including 70 patients fulfilling the revised Ghent criteria clinically and 14 patients having aortopathy and systemic score ≥5 of Marfan syndrome without ectopia lentis.3 Among them, 2 patients were clinically diagnosed with Loeys–Dietz syndrome. Whole exome sequencing was conducted using Japan’s Initiative on Rare and Undiagnosed Diseases in Pediatrics4 research for 17 patients from September 2015 to September 2016, and hybridization capture-based gene-panel testing for aortopathies including FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, ACTA2, MYH11, MYLK, and COL3A1, was conducted at the Kazusa DNA Research Institute (Chiba, Japan) for 67 patients from April 2018 to September 2020. Simultaneous processing of DNA samples in a single sequencing run was not guaranteed. This study was approved by the institutional ethics committee (G-1538).The mean age at the time of genetic testing was 28.6±17.3 years and 54 were male (64.3%). Pathogenic or likely pathogenic single nucleotide variants or short indels were identified in 69 patients (82.1%): FBN1 (n=64), TGFBR1 (n=3), TGFBR2 (n=1), and SKI (n=1: c.539C>T, p.Thr180Met). SKI is the causative gene of Shprintzen-Goldberg syndrome, characterized by craniosynostosis with marfanoid habitus, and recently variants affecting Thr180 have been reported to cause aortopathy.5 Next, we simply predicted CNVs via visual review of the depth of coverage (doc) patterns using the Integrative Genomics Viewer with the aid of Excel 2-dimensional line chart for all patients (Figure [A] and [B]). Three independent inspectors called seven suspected heterozygous intragenic (multi-)exon deletions in FBN1 (n=6) and TGFB2 (n=1). They had a patient/control doc ratio of ≈0.5 for each targeted exon.Download figureDownload PowerPointFigure. Copy number variation analysis for syndromic aortopathies.A–C, Representative results of copy number variation (CNV) screening obtained from patient no. 1066. A, Visual screening for CNVs by comparing integrative genomics viewer read coverage tracks: top, a representative normal depth of coverage (doc) pattern; bottom, low-height doc patterns in the red enclosed part. B, top, Read depth (RD) plot throughout the FBN1 gene of 5 samples with close collection dates. Bottom, RD for each was normalized by dividing by the average of all RDs of the gene. Arrow indicates a marked decline in CNV-suspected region. C, Array-comparative genomic hybridization (CGH). The horizontal axis represents the nucleotide position. The vertical axis represents log2 (case/reference signal intensities on array-CGH). Dots with log2 (case/reference signal intensities) >0 are indicated in blue, and those <0 are indicated in red. The red-shaded area is a significant CNV region called by the Agilent CytoGenomics software. D, Overview of the CNVs identified in this study. In patient no. 655, exon 4 to exon 8, the last coding exon of the TGFB2 gene, was heterozygously deleted. E, Summary of genetic tests. VUS indicates variant of unknown significance.To confirm the genetic alterations and identify the breakpoint locations, we conducted an Agilent custom-designed oligonucleotide array-comparative genomic hybridization (array-CGH; average probe interval 185 bp) analysis in the 7 patients, which covered 10 genes described above and detected the suspicious regions (Figure [C]). Sequencing analysis using primers flanking the breakpoint revealed the details of each breakpoint located in an intron; the heterozygous deletion ranged from 1.0 to 26.2 kb with insertion sequences (Figure [D]). In patient no. 1038, the mutant allele had a 1451-bp deletion, with an insertion of ≈90 to 100 bp, including poly-thymine (poly-T)70-80 tracts. The involvement of the Alu-associated mechanism was highly likely at the 3′ breakpoint region within intron 40 (chr15:48 464 229–48 464 556), which composed of a thymine (T)-rich region and the subsequent downstream 287 bp having 83% homology with the inverted Alu consensus sequence.The clinical significance of CNV in hereditary diseases is becoming increasingly clear. Multiplex ligation-dependent probe amplification and array-CGH are the gold standard methods in genotyping CNV, but still time-consuming and expensive approach if the number of exons and genes of interest becomes larger. Systematic, accurate exome/panel sequence-based CNV assessment is still challenging, and thus we simply performed visual comparison of the doc patterns using the Integrative Genomics Viewer, because the number of known genes that predispose a carrier to syndromic aortopathy is still limited. Even with such simple methodology, we were able to identify 7 exonic CNVs in patients with syndromic aortopathy, which accounted for 46.7% of the causative variants in 15 patients having no pathogenic or likely pathogenic single nucleotide variants and short indels (Figure [E]). It might still be possible to have overlooked pathological CNVs, but our study definitely reinforced the importance of exonic CNV analysis for syndromic aortopathies. The incidence rate might be more than previously reported, and further investigation is needed to identify exonic CNVs in targeted sequencing data generated in daily medical practice. If routine next-generation sequencing is negative in such patients, CNV analysis should be incorporated into the clinical diagnostic care and our simple methodology can be used for this purpose at least as a first-tier testing before conventional array-CGH. The CNV breakpoint sequences are valuable for the genetic diagnosis and counseling for suspected but clinically unaffected family members, as well as the identification of the disease mechanism.AcknowledgmentsWe thank all the individuals who participated in this study and whose contributions made this work possible.Sources of FundingThis work was supported by a Grant-in-Aid for Research on Rare and Intractable Diseases from Japan Agency for Medical Research and Development (AMED) to Dr Komuro (JP20ek0109487) and partially supported by a grant from the Initiative on Rare and Undiagnosed Diseases in Pediatrics (IRUD-P) project (16ek0109166h0002) from AMED.Disclosures None.Footnotes*N. Takeda and R. Inuzuka contributed equally.For Sources of Funding and Disclosures, see page 531.Correspondence to: Issei Komuro, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Email [email protected]ac.jpNorifumi Takeda, MD, PhD, Department of Cardiovascular Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Email [email protected]ac.jp