TY - JOUR
T1 - Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients
AU - Gravesteijn, Gido
AU - Dauwerse, Johannes G.
AU - Overzier, Maurice
AU - Brouwer, Gwendolyn
AU - Hegeman, Ingrid
AU - Mulder, Aat A.
AU - Baas, Frank
AU - Kruit, Mark C.
AU - Terwindt, Gisela M.
AU - van Duinen, Sjoerd G.
AU - Jost, Carolina R.
AU - Aartsma-Rus, Annemieke
AU - Lesnik Oberstein, Saskia A. J.
AU - Rutten, Julie W.
PY - 2020/7/21
Y1 - 2020/7/21
N2 - CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.
AB - CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.
KW - CADASIL
KW - NOTCH3
KW - exon skipping
KW - protein aggregation
KW - small vessel disease
UR - http://www.scopus.com/inward/record.url?scp=85082740741&partnerID=8YFLogxK
U2 - https://doi.org/10.1093/hmg/ddz285
DO - https://doi.org/10.1093/hmg/ddz285
M3 - Article
C2 - 31960911
SN - 0964-6906
VL - 29
SP - 1853
EP - 1863
JO - Human Molecular Genetics
JF - Human Molecular Genetics
IS - 11
ER -