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Low-level somatic convoluted indel variants in C-terminal region of TEK revealed by deep sequencing on patients with vascular malformations

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction
Somatic pathogenic variants in the epithelial-specific protein receptor tyrosine kinase gene TEK are known to cause sporadic venous malformations, characterized by small skin lesions consisting of dilated vascular spaces with localized tissue-specific manifestations. Somatic variants with low variant allelic fractions (VAF) are challenging to be detected without application of high-depth sequencing tests.

Case Presentation
We report two unrelated individuals affected with sporadic vascular anomalies. The first individual is a 5-year-old female with a facial arteriovenous malformation, and an affected fresh tissue biopsy was submitted for analysis. The second individual is a 15-year-old male presenting with a left temporal mass demonstrating evidence of venous malformation in the brain, and a formalin-fixed paraffin-embedded tissue sample was analyzed.

Diagnostic Workup
Diagnostic specimens were submitted for DNA extraction followed by a targeted clinical deep sequencing of a set of genes associated with disorders of somatic mosaicism and related conditions. The read depth of the sequencing panel for these two cases reached beyond 4000x. Multiple variant callers including GATK, FreeBayes, Mutect, Varscan and Vardict were employed, and data analysis was supported by a customized bioinformatic pipeline (Genoox Annotation version 77). Variant assessment was subject to the guidelines from the American College of Medical Genetics and Genomics/Association for Molecular Pathology as well as an internal variant assessment algorithm. In the first case, we identified a likely pathogenic variant in the last exon of TEK (NM_000459.5): c.3314_3317delinsTGAGAAGTTTACTTATGCAGGAATTAT (p.Thr1105Metfs*47). Another similar variant located 6-bp apart, TEK (NM_000459.5): c.3323_3324insCGTGAATACCA CGCTTTATGAGAAGTAGTA (p.Tyr1108_Glu1109insValAsnThrThrLeuTyrGluLys*) was detected in the second case. These two complex insertion-deletion (indel) variants were identified at low VAF (<10%), consistent with somatic origin. Neither variant, to our knowledge, has been reported in the medical literature and is absent from the general population (Genome Aggregation Database v4.1.0). Of note, due to the size and complexity of these variants and the use of short-read sequencing methods, alignment of these variants led to our variant calling pipelines representing this single event with multiple variant calls in each case. This complexity makes the VAF estimate in this specimen unreliable and likely underestimated. None of these variant calls accurately represented the complexity of these variants and aligned reads showed multiple representations in the Integrated Genomics Viewer (IGV). Ultimately, to support a proper HGVS representation of these complex events (which included inserted, partially-duplicated, and deleted sequence) required manual review and intervention.

 

Discussion
Two complex indel variants reside in the C-terminus of TEK, a critical inhibitory loop for the tyrosine kinase function. Other frameshift and indel variants in this region have been reported in multiple patients with venous malformations, suggesting a potential mutational hotspot. Several studies have suggested that truncating variants in this region may result in conformational changes in the inhibitory loop, leading to increased receptor autophosphorylation. 

Conclusion
These two cases represent two complex, and previously not described, pathogenic indels in the last exon of TEK that may be missed due to their intermediate size (27-30bp) and low VAFs that are further skewed by their complexity and alignments of short read sequencing. Furthermore, recurrent observations of complex variants detected in the C-terminal region of TEK from patients with VMCM emphasize that this genomic region may require careful assessment and manual inspection of the sequence.

 

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