Congruence between NOTCH3 mutations and GOM in 131 CADASIL patients

Abstract

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary subcortical vascular dementia. It is caused by mutations in NOTCH3 gene, which encodes a large transmembrane receptor Notch3. The key pathological finding is the accumulation of granular osmiophilic material (GOM), which contains extracellular domains of Notch3, on degenerating vascular smooth muscle cells (VSMCs). GOM has been considered specifically diagnostic for CADASIL, but the reports on the sensitivity of detecting GOM in patients' skin biopsy have been contradictory. To solve this contradiction, we performed a retrospective investigation of 131 Finnish, Swedish and French CADASIL patients, who had been adequately examined for both NOTCH3 mutation and presence of GOM. The patients were examined according to the diagnostic practice in each country. NOTCH3 mutations were assessed by restriction enzyme analysis of specific mutations or by sequence analysis. Presence of GOM was examined by electron microscopy (EM) in skin biopsies. Biopsies of 26 mutation-negative relatives from CADASIL families served as the controls. GOM was detected in all 131 mutation positive patients. Altogether our patients had 34 different pathogenic mutations which included three novel point mutations (p.Cys67Ser, p.Cys251Tyr and p.Tyr1069Cys) and a novel duplication (p.Glu434_Leu436dup). The detection of GOM by EM in skin biopsies was a highly reliable diagnostic method: in this cohort the congruence between NOTCH3 mutations and presence of GOM was 100%. However, due to the retrospective nature of this study, exact figure for sensitivity cannot be determined, but it would require a prospective study to exclude possible selection bias. The identification of a pathogenic NOTCH3 mutation is an indisputable evidence for CADASIL, but demonstration of GOM provides a cost-effective guide for estimating how far one should proceed with the extensive search for a new or an uncommon mutations among the presently known over 170 different NOTCH3 gene defects. The diagnostic skin biopsy should include the border zone between deep dermis and upper subcutis, where small arterial vessels of correct size are located. Detection of GOM requires technically adequate biopsies and distinction of true GOM from fallacious deposits. If GOM is not found in the first vessel or biopsy, other vessels or additional biopsies should be examined.

Figure 1

A small arteriole (left) and venule (right) in the lower dermis of the skin biopsy from a Finnish CADASIL patient with p.Arg133Cys mutation. Note the somewhat thicker wall and lamina elastica interna (dark blue spots beneath the endothelium) in the arteriole. Toluidine blue-stained semithin epon section.

Figure 2

(A) The same vein as in Fig. 1 with no GOM. (B) A vein from deep dermis with one definite GOM deposit (arrow) shown at a higher magnification in the inset. (C) The arteriole in Fig. 1 with several GOM deposits (five shown with arrows), one marked with asterisk is shown at a higher magnification in the inset. Note the characteristic pinocytotic vesicles at the VSMC plasma membrane beneath the GOM. L = lumen, E = endothelium, M = vascular smooth muscle cell.

Figure 3

Fallacious deposits which may lead the electron microscopist astray. (A) True GOM with a somewhat exceptional mushroom-like form (arrows). Note the characteristic finely granular structure of GOM. (B) Fragments of elastica interna (ELA) and granular fibrillin network (F) in the widened subendothelial space. (C) Granular debris of unknown origin (asterisk). (D) Similar granular material as in C (arrow) with a misleading localization in a cove of VSMCs. (E and F) Small clumps of cell debris of different composition (arrows), possibly from degenerated cells. L = lumen, E = endothelium, M = vascular smooth muscle cell.

Figure 4

A small GOM deposit is in a deep indentation on a VSMC in a dermal arteriole from a 19-year-old CADASIL patient with p.Arg133Cys mutation. Note the pinocytotic vesicles.