Disruption of a miR-29 binding site leading to COL4A1 upregulation causes PADMAL

OBJECTIVE: Cerebral small vessel disease (cSVD) is an heterogeneous group of disorders. Screening of known cSVD genes identifies the causative mutation in less than 15% of familial cSVD cases. We sought to identify novel causes of cSVD.

METHODS: We used linkage analysis and exome sequencing to identify the causal mutation in a French cSVD family. The identified candidate gene was then screened in 202 cSVD unrelated probands, including one proband from the first reported Pontine Autosomal Dominant MicroAngiopathy with Leukoencephalopathy (PADMAL) family. Sanger sequencing was used to confirm variants in all mutated probands and analyze their segregation in probands' relatives. Mutation consequences were assessed with luciferase reporter assays and RT-qPCR.

RESULTS: A candidate heterozygous variant located in a predicted miR-29 microRNA binding site, within the 3'UTR region of COL4A1, was identified in the large French cSVD family. Five additional unrelated probands, including the PADMAL proband, harbored heterozygous variants in this microRNA binding site. Variants cosegregated with the affected phenotype and cumulative LOD score reached 6.03, establishing linkage to this locus. A highly significant difference was observed when comparing the number of variants within this binding site in cases and controls (p=1.77 x 10E-12). RT-qPCR analyses of patients' primary fibroblasts and luciferase reporter assays strongly favor an upregulation of COL4A1 mediated by disruption of miR-29 binding to its target site. MRI features were characterized by the presence of multiple pontine infarcts in all symptomatic mutation carriers.

INTERPRETATION: Mutations upregulating COL4A1 expression lead to PADMAL, a severe early-onset ischaemic cSVD, distinct from the various phenotypes associated with COL4A1 missense glycine mutations. This article is protected by copyright. All rights reserved.