Disruption of a miR-29 binding site leading to COL4A1 upregulation causes pontine autosomal dominant microangiopathy with leukoencephalopathy.
OBJECTIVE
Cerebral small vessel disease (cSVD) is a heterogeneous group of disorders. Screening of known cSVD genes identifies the causative mutation in <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 1 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 real-time quantitative polymerase chain reaction (RT-qPCR).


RESULTS
A candidate heterozygous variant located in a predicted miR-29 microRNA binding site, within the 3' untranslated 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 logarithm of odds 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 × 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. Magnetic resonance imaging features were characterized by the presence of multiple pontine infarcts in all symptomatic mutation carriers.


CONCLUSIONS
Mutations upregulating COL4A1 expression lead to PADMAL, a severe early onset ischemic cSVD, distinct from the various phenotypes associated with COL4A1 missense glycine mutations. Ann Neurol 2016;80:741-753.
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.
Edgard
E
Verdura
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.|AP-HP Lariboisière Neurology Department, CERVCO Reference Center for Rare Vascular Diseases of the Eye and Brain, Paris, France.
Dominique
D
Hervé
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.
Françoise
F
Bergametti
Neurology department, CHRU of Lille, Lille 2 University, Inserm U1171, Lille, France.
Clémence
C
Jacquet
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.
Typhaine
T
Morvan
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.|AP-HP Lariboisière Molecular Genetics Department, CERVCO Reference Center for Rare Vascular Diseases of the Eye and Brain, Paris, France.
Carol
C
Prieto-Morin
Neurology Department, Saint Philibert Hospital, Lomme, France.
Alexandre
A
Mackowiak
Neurology Department, Gonesse Hospital, Gonesse, France.
Eric
E
Manchon
Neurology Department, Henri Mondor Hospital, Creteil, France.
Hassan
H
Hosseini
Neurology department, CHRU of Lille, Lille 2 University, Inserm U1171, Lille, France.
Charlotte
C
Cordonnier
Neurology Department, Valenciennes Hospital, Valenciennes, France.
Isabelle
I
Girard-Buttaz
Neurology Department, Coulommiers Hospital, Coulommiers, France.
Sophie
S
Rosenstingl
Institute of Neuropathology, University Medical Center, Hamburg, Germany.
Christian
C
Hagel
Department of Neurology, University Hospital Schleswig-Holstein, Kiel, Germany.
Gregor
G
Kuhlenbaümer
Neurology Department, Agen Hospital, Agen, France.
Elena
E
Leca-Radu
CMAbio3, Applied Microscopy and Biology Center, University of Caen, Caen, France.
Didier
D
Goux
Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Lauren
L
Fleming
Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Tom
T
Van Agtmael
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.|AP-HP Lariboisière Neurology Department, CERVCO Reference Center for Rare Vascular Diseases of the Eye and Brain, Paris, France.
Hugues
H
Chabriat
Pathology Department, CHRU of Caen, University of Caen Normandy, Inserm, U1075.
Françoise
F
Chapon
Inserm U1161, Genetics and Physiopathology of Cerebrovascular Diseases, Paris, France.|Inserm U1161, Paris7 Diderot University, Sorbonne Paris Cité, Paris, France.|AP-HP Lariboisière Molecular Genetics Department, CERVCO Reference Center for Rare Vascular Diseases of the Eye and Brain, Paris, France.
Elisabeth
E
Tournier-Lasserve
D017668:Age of Onset; D059345:Cerebral Small Vessel Diseases / Q000000981:diagnostic imaging / Q000235:genetics / Q000503:physiopathology; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism*; D059472:Exome; D005260:Female; D005602:France; D008040:Genetic Linkage; D006801:Humans; D056784:Leukoencephalopathies / Q000000981:diagnostic imaging / Q000235:genetics / Q000503:physiopathology; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D011149:Pons / Q000000981:diagnostic imaging*; D011485:Protein Binding; D015854:Up-Regulation
C547475:COL4A1 protein, human; D024141:Collagen Type IV; C532045:MIRN29a microRNA, human; D035683:MicroRNAs