Genetic Alterations Underlie Hereditary Arteriovenous Malformation Pathogenesis

Abstract

Arteriovenous malformations (AVMs) are rare, dangerous vascular malformations consisting of abnormal direct connections between arteries and veins. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant syndrome characterized by AVMs on the skin and internal organs. Patients inherit a heterozygous loss-of-function mutation in ENG, ACVRL1, or SMAD4. Haploinsufficiency has been proposed as the primary disease mechanism, but this does not explain why AVMs are focal, discrete lesions. In contrast, a Knudsonian genetic two-hit model proposes that AVM pathogenesis requires local biallelic loss of an HHT gene due to somatic mutation. Biallelic loss of ENG or ACVRL1 was previously identified in half of HHT-associated skin AVMs, but whether all HHT-associated AVMs require biallelic loss of HHT genes remains unknown. We sequenced DNA from HHT-associated liver, lung, and brain AVMs using targeted short-read sequencing. We found somatic mutations likely to cause loss-of-function in the gene of the germline mutation (ENG or ACVRL1) in AVMs, indicating that internal organ AVMs are also seeded by somatic biallelic loss-of-function. In AVMs where we did not identify a somatic mutation, we analyzed genome wide single nucleotide polymorphism (SNP) genotyping data with the program Mosaic Chromosomal Alterations (MoChA) to identify large regions of loss-of-heterozygosity. We found loss-of-heterozygosity over the gene of the germline mutation in skin and liver AVMs, identifying loss-of-heterozygosity as another mechanism of somatic loss of HHT genes in HHT-associated AVMs. In a craniofacial AVM from a patient with a germline SMAD4 mutation, we found two somatic loss-of-function events in SMAD4, a point mutation and loss-of-heterozygosity. Using long read sequencing and SNP genotyping, we showed that each event caused biallelic loss of SMAD4 in the AVM, evidence that biallelic loss of SMAD4 also seeds HHT-associated AVMs. Altogether, these results indicate that biallelic genetic inactivation of HHT genes is likely a universal requirement for HHT-associated vascular malformation pathogenesis.