Supplementary Material for: FAM222B Is Not a Likely Novel Candidate Gene for Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, <i>CCM1</i><i>(KRIT1)</i>, <i>CCM2</i><i>(MGC4607)</i>, and <i>CCM3</i><i>(PDCD10)</i> in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further <i>CCM</i> gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, <i>FAM222B</i><i>(C17orf63)</i>, through exome sequencing of CCM patients mutation-negative for <i>CCM1-3</i>. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in <i>fam222bb</i>/<i>fam222ba</i> double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against <i>FAM222B</i>. These observations led to the assumption that aberrant <i>FAM222B</i> is not involved in the formation of CCMs.