Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients.

Neven Maksemous,Lyn R Griffiths,Larisa M Haupt,Robert A Smith

doi:10.1186/s40246-016-0093-z

Abstract

BackgroundCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing.ResultsOur custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing.ConclusionsNGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-016-0093-z) contains supplementary material, which is available to authorized users.

Highlights

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults
Sequencing data analysis Comprehensive screening for NOTCH3 using the AmpliSeq Custom next-generation sequencing (NGS) panel [7] (Thermo Fisher Scientific, Scoresby, Victoria, Australia) for targeted gene sequencing was conducted on 44 patients, previously screened for standard sequencing exons (3 and 4) and/or (2,11, 18 and 19) by Sanger sequencing (SS) and classified as being negative for known mutations
Nine notable genetic variants were identified in 10 patients (22.7 %) out of the 44 subjects: five novel potential mutations (NOTCH3:NM_000435:exon4:c.416A>T:p.D139V, exon1 1:c.1791C>G:p.C597W and c.1820G>A: p.R607H, exon18: c.2929T>G,: p.C977G; and exon20: c.3317A>G: p.Y1106C); three previously reported disease-causing missense mutations (NOTCH3:NM_000435:exon4:c.547T>Cp.C183R [8], exon 6:c.994C>T, p.R332C [9, 10]; and exon9: c.1394A>G: p.Y465R [11]) and one novel synonymous genetic variant in NOTCH3:NM_000435:exon16:c.2439G>A: p.E813E [Tables 1 and 2]

Summary

Introduction

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. The stroke syndrome CADASIL [MIM 125310] (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) disorder results in neuronal white matter abnormalities and is characterised by a variety of symptoms including, vascular degeneration, recurrent subcortical ischaemic strokes, progressive cognitive decline, dementia, migraine with aura (22 % of patients) and premature death [1]. We have performed targeted gene sequencing using a custom fivegene NGS panel [7], encompassing the coding sequences, 20–100 bp exon/intron boundaries and the 5′ and 3′UTR regions of NOTCH3 in 44 patients

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