First interchromosomal insertion in a patient with cerebral and spinal cavernous malformations

Abstract

Autosomal dominant cerebral cavernous malformations (ccM) are leaky vascular lesions that can cause epileptic seizures and stroke-like symptoms. Germline mutations in either CCM1, CCM2 or CCM3 are found in the majority of patients with multiple CCMs or a positive family history. Recently, the first copy number neutral inversion in CCM2 has been identified by whole genome sequencing in an apparently mutation-negative CCM family. We here asked the question whether further structural genomic rearrangements can be detected within nGS gene panel data of unsolved ccM cases. Hybrid capture nGS data of eight index patients without a pathogenic single nucleotide, indel or copy number variant were analyzed using two bioinformatics pipelines. In a 58-year-old male with multiple CCMs in his brain and spinal cord, we identified a 294 kb insertion within the coding sequence of CCM2. fine mapping of the breakpoints, molecular cytogenetic studies, and multiplex ligation-dependent probe amplification verified that the structural variation was an inverted unbalanced insertion that originated from 1p12-p11.2. As this rearrangement disrupts exon 6 of CCM2 on 7p13, it was classified as pathogenic. Our study demonstrates that efforts to detect structural variations in known disease genes increase the diagnostic sensitivity of genetic analyses for well-defined Mendelian disorders. Cerebral cavernous malformations (CCM; MIM: 116860, 603284, 603285) are irregular clusters of enlarged and thin-walled vessels that can present as sporadic or autosomal dominant cerebrovascular disease. Aside from epileptic seizures and headaches, CCM patients may present with stroke-like symptoms due to chronic or acute bleeding events 1. Pathogenic germline variants have been identified in CCM1 (also known as KRIT1) 2,3 , CCM2 4,5 , and CCM3 (PDCD10) 6. The mutational spectrum primarily includes nonsense, frameshift, splice, and copy number variants (CNVs). 282 unique CCM1, 84 CCM2 and 75 CCM3 variants are classified as disease-causing in the Human Gene Mutation Database (HGMD Professional 2019.3) 7. Depending on the inclusion criteria for genetic analyses, mutation detection rates of 87 to 98% have been reported for familial CCM cases and up to 60% for sporadic ones 8-11. The mutation detection rate in the latter group could even be higher if patients with associated developmental venous anomalies or a history of radiotherapy to the brain were excluded 1,12. Although pathogenic variants in a yet unknown CCM4 candidate gene have been discussed for unresolved cases 13 , we recently were able to identify the first copy number neutral inversion in CCM2 in an apparently mutation-negative CCM family by whole genome sequencing (WGS) 14. Disease-causing structural variants (SVs) may explain a part of the "missing heritability" in rare diseases 15. SVs are defined as structural and quantitative chromosomal rearrangements that compromise cytogenetically visible and submicroscopic variants 16,17. They contribute to phenotypic variation but can also cause human diseas...

Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion

“…Nonetheless, no causative variant is identified in 2%–13% of familial CCM cases by current genetic analyses ( Spiegler et al, 2018b ). Apart from structural variants ( Spiegler et al, 2018a ; Pilz et al, 2020 ) and somatic mosaicism ( McDonald et al, 2014 ), the here identified CCM1 TSS deletion highlights that variants in regulatory regions that are not always analyzed in routine diagnostics may account for part of the missing heritability in CCM disease.…”

Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM Genes

“…The detection of complex SVs is hard or even impossible with short-read gene panel sequencing. Since the identification of an interchromosomal insertion and an inversion in CCM2 [ 8 , 9 ] suggests that SVs must be considered as a possible cause of familial CCM disease, we wanted to test whether long-read sequencing could accurately detect these variants. Therefore, we reanalyzed a heterozygous 24 kb inversion on chromosome 7 ( Figure 4 a), which covers the first coding exon of CCM2 [ 8 ], with long-read sequencing ( Figure 4 b; Table S3 ).…”

“…In the next step, we re-analyzed a known heterozygous interchromosomal insertion of chromosome 1 material into the coding region of CCM2 ( Figure 5 a). This variant which we had previously called from short read gene panel sequencing data with the SureCall software, was an ideal positive control for our approach because it already had been extensively characterized and confirmed by FISH [ 9 ]. Long-read sequencing of DNA with this variant ( Table S3 ) generated single reads longer than 20 kb.…”

“…For example, they can hardly identify the precise breakpoints of CNVs, pathogenic variants in non-coding regions, and complex structural variants (SVs), e.g., inversions or interchromosomal insertions. This is particularly relevant since all these types of genetic variation have been described for CCM patients in the recent literature [ 6 , 7 , 8 , 9 ]. In the context of analyses for CNVs segregating within CCM families, cost-effectiveness is also an issue.…”

3D Digital Anatomic Angioarchitecture of the Rat Spinal Cord: A Synchrotron Radiation Micro-CT Study

“…A wide range of spinal cord-related neurovascular diseases and other pathological processes are associated with vascular alterations (Wu et al, 2008 ; Heldner et al, 2012 ). Such as the cavernous hemangiomas in the spinal cord consist of abnormal blood vessels with poor blood flow (Pilz et al, 2020 ). The spinal dural arteriovenous (AV) fistulas are the most commonly encountered vascular malformation of the spinal cord and still underdiagnosed entities (Takai et al, 2020 ).…”