The first deep intronic mutation in the NOTCH3 gene in a family with late-onset CADASIL

Neurobiology of Aging 34 (2013) 2234.e9e2234.e12

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The first deep intronic mutation in the NOTCH3 gene in a family with late-onset CADASIL Silvia Bianchi a, Maria Teresa Dotti a, Gian Nicola Gallus a, Camilla D’Eramo a, Ilaria Di Donato a, Livia Bernardi b, Raffaele Maletta b, Gianfranco Puccio b, Amalia C. Bruni b, Antonio Federico a, * a b

Department of Medical, Surgical and Neurological Sciences, University of Siena, Siena, Italy Regional Neurogenetic Centre, ASPCZ, Lamezia Terme, Catanzaro, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 July 2012 Received in revised form 2 March 2013 Accepted 11 March 2013 Available online 12 April 2013

CADASIL is the most prominent inherited form of vascular dementia. The main clinical features include migraine with aura, stroke, mood disturbances, and cognitive decline, with a mid-life (30s-60s) adult onset. Genetic testing is the gold standard for the diagnosis. CADASIL is caused mostly by missense mutations in the NOTCH3 gene, invariably involving a cysteine residue. Only a couple of splice site mutations have been reported. In a few pathologically defined patients, genetic mutations remain unidentified. We report a family with late-onset CADASIL phenotype carrying a novel intronic deletion in the NOTCH3 gene (c.341-26_24delAAC). Transcript analysis revealed a splicing alteration, with the complete intron 3 retention. The insertion was in-frame and encoded an extra 25 amino acids, including 1 cysteine. This is the first report of an aberrant splicing event of the NOTCH3 gene associated with a mutation far away from the canonical splice site. Our finding suggests that the assays used to evaluate splicing should be mandatory in the diagnostic setting of genetically undefined CADASIL cases. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Vascular dementia NOTCH3 gene Intronic deletion Splicing alteration

1. Introduction CADASIL is the most prominent inherited form of small vessel disease in adults (Chabriat et al., 2009). The main clinical features include migraine with aura, subcortical ischemic events, mood disturbances, and cognitive decline. The disease, usually manifesting before the age of 60 years, has a progressive course, with severe disability and dementia in the advanced stage (Chabriat et al., 2009). However, the CADASIL phenotype is highly variable, even within families (Dichgans et al., 1998). Magnetic resonance imaging (MRI) of the brain shows widespread white matter lesions, frequently extending to the anterior pole of the temporal lobe, and associated with lacunar infarcts (Dichgans et al., 1998). The pathologic hallmark of the disease is the presence of granular osmiophilic material (GOM) on electron microscope examination of the smooth muscle cells in cerebral and extracerebral vessels, including dermal arterioles (Chabriat et al., 2009). Thus, * Corresponding author at: Dipartimento Medicina, Chirurgia e Neuroscienze, Università di Siena, Viale Bracci, 53100 Siena, Italy. Tel.: þ39 0577 585760; fax: þ39 0577 40327. E-mail address: [email protected] (A. Federico). 0197-4580/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.03.005

skin biopsy is considered a highly specific, albeit not entirely sensitive, disease marker (Malandrini et al., 2007). CADASIL is caused by mutations in the NOTCH3 gene, and genetic testing is the gold standard for diagnosis (Joutel et al., 1997). However, in a small percentage of GOM-positive CADASIL patients, the exact identification of NOTCH3 mutations is not possible (Peters et al., 2005). Most of the approximately 200 pathogenic mutations identified to date are missense, with some deletion or insertion and only 2 splicing alterations (http:// chromium.liacs.nl/LOVD2/home.php?select_db¼NOTCH3; Joutel et al., 2000; Saiki et al., 2006). In human genetic disease, splicing errors account for about 50% of all point mutations (Lopez-Bigas et al., 2005). Most of these sequence variations are due to a mutation in the splice site, but an increasing number of splicing mutations are located in exons and introns far from the splice junction (Ward and Cooper, 2010). In fact, many cis-acting intronic and exonic sequences determine the precision with which introns are recognized and removed. Among these, the branch point sequence (BPS) is the best characterized (Gao et al., 2008). We report an Italian family with late-onset typical CADASIL symptoms carrying a novel splicing mutation of the NOTCH3 gene

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due to an intron mutation far from canonical splice sites, which generates an aberrant splicing event with intron 3 retention. 2. Methods 2.1. Patients Table 1 summarizes the clinical and genetic characteristics of our patients. The index patient (III-5; family pedigree depicted in the Fig. 1B) is a 73-year-old man first observed at the age of 66 years because of a 5-year history of progressive cognitive decline. His medical history was unremarkable until the age of 61, when short-term memory impairment, mental slowing, and personality changes were noticed. His Mini-Mental State Examination (MMSE) score was 25.5 (cognitive impairment defined as MMSE 23). Brain MRI showed widespread T2-weighted and FLAIR (Fluid Attenuated Inversion Recovery) hyperintense lesions mainly involving the periventricular white matter, the anterior part of temporal lobes, basal ganglia, thalamus, and corpus callosum (Fig. 1A). The clinical picture slowly deteriorated toward a pseudo bulbar palsy. The patient’s mother (II-1) died at the age of 77 for unknown causes;, the patient’s older brother (III-1) died at the age of 67 of sudden death, and another brother (III-3) died at 66 years of a stroke. The 80-year-old sister (III-10) experienced severe cognitive impairment from the age of 63. Computed tomography of the brain evidenced multiple white matter lesions and diffuse leukoencephalopathy. Patient III-14 had a history of migraine, hypertension, hypercholesterolemia, and hypertriglyceridemia. At the age of 65 years, he acutely presented with 2 episodes of cerebral ischemia, both occurring within 1 week, with left hemiparesis and global aphasia. The course of the disease worsened, leading to a condition of pseudo bulbar palsy. He died at age 78 years. The proband’s younger son (IV-8), aged 35 years, was clinically asymptomatic. MRI of the brain revealed a small white matter hyperintensity in the right hemisphere. The other examined family members (III-8, III-12, and IV-9) did not show clinical or MRI abnormalities. 2.2. Genetic analysis 2.2.1. Mutation detection For the detection of NOTCH3 gene mutations in genomic DNA, DNA of the proband and family members was extracted using routine procedures from peripheral blood lymphocytes. Exons 2 to 24 (with exon/intron boundaries) and complete introns 3, 5, 7, 9, 11, 13, 14, 18, 21, and 22 were sequenced. Polymerase chain reaction (PCR) conditions and primer sequences are available upon request. The resulting amplicons were visualized on a 2% agarose gel, and used as template for sequencing with the Big-Dye terminator cycle sequencing kit (Applied Byosistems) and an ABI 3100 automated sequencer. The PCR products containing the c.341-26_24delAAC in the heterozygous state was then cloned by the StrataClone PCR Cloning kit (Stratagene) following the manufacturer’s instructions, and the two alleles were separately sequenced.

2.2.2. RNA (cDNA synthesis, amplification and sequencing) Total RNA was extracted using a RNeasy Mini kit (Qiagen) from cultured fibroblasts. First-strand cDNA was reverse transcribed from 1 mg of poly (A)þ RNA using the ImProm-II Reverse Transcriptase (Promega). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified as a control. Amplification of the region of exons 2 to 4 (primers Afw: 50 -gcaaatggaggtcgttgcac-30 and Brv: 50 -atccacgtcgcttcggcag-30 ) and the region of exons 2 to 5 (primers Afw: 50 -gcaaatggaggtcgttgcac-30 and Crv: 50 -ggcactggcagttataggtg-30 ) of the NOTCH3 cDNA was carried out. The resulting amplicons were visualized on a 2% agarose gel, purified from the gel using the QIAquick PCR purification kit (Qiagen) and used as template for sequencing in both senses with Big-dye terminator cycle sequencing reaction kit (Applied Biosystems) and an ABI 3100 automated sequencer. 2.2.3. Mutation nomenclature Mutation designation is done according to the official mutation nomenclature (http://www.hgvs.org/mutnomen/). The reference accession numbers were GenBank NM_000435.2 for cDNA, and NG_009819 for genomic DNA. 2.3. In silico analysis of intron 3 DNA sequence analysis of wild-type and mutated sequence of intron 3 was performed using Human Splicing Finder version 2.4.1(http://www.umd.be/HSF/), Branch Site Analyzer (http://ibis. tau.ac.il/ssat/BranchSite.htm) and SVM-BP finder (http:// regulatorygenomics.upf.edu/SVM_BP/). 3. Results DNA molecular analysis of the proband was performed for exons 2 to 24, exon/intron boundaries, and some entire introns of the NOTCH3 gene, including intron 3. A previously undescribed heterozygous deletion mutation was identified in intron 3 (c.34126_24delAAC); no other changes in the coding sequence or canonical splice sites were found. The c.341-26_24delAAC mutation was also detected in 1 asymptomatic and 2 affected relatives (Table 1), whereas it was absent in all of the unaffected family members tested (III-8, III-12, and IV-9). Furthermore, this mutation was not found in 120 healthy subjects, and it was not present in the dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) and 1000 Genomes (http://browser.1000genomes.org/index.html) databases. Genetic and ultrastructural studies (Table 1) showed segregation of the c.341-26_24delAAC mutation in this family. In silico analysis was performed to evaluate the potential for defective splicing of the mutation detected. The software used for BPS prediction indicated that the 3-nucleotide deletion is part of 2 putative BPS (Fig. 1C; Supplementary material: eTable 1). To determine the effect of this mutation on NOTCH3 pre-mRNA splicing, total RNA, extracted from the cultured fibroblasts from III-5, IV-8, IV-9, and from 1 control subject, was reverse transcribed and amplified using primers specifically designed to detect aberrant splicing involving either exon 3 or exon 4. Two bands were seen upon gel electrophoresis of

Table 1 Main clinical, genetic and MRI features of family members ID patient

Age (y)

Clinical features

MRI findings

Skin biopsy

c.341-26_24delAAC

III-3 III-5 III-10 III-14 IV-8

O: >60; D: 66 O: 61; C: 73 O: 63; C: 80 O: 65; D: 78 C: 35

Progressive cognitive impairment Progressive cognitive impairment Progressive cognitive impairment Migraine, stroke, progressive cognitive impairment Asymptomatic

Diffuse leukoencephalopathy Diffuse leukoencephalopathy Diffuse leukoencephalopathy Diffuse leukoencephalopathy Small focal white matter hyperintensities

NP GOM NP NP GOM

ND þ þ þ þ

Key: C, current age; D, age at death; ND, not determined; NP, not performed; O, age at onset.

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Fig. 1. (A) Magnetic resonance imaging (MRI) of the brain of patient III-5: Diffuse and confluent hyperintense lesions in the periventricular and deep white matter (1) on an axial FLAIR image. White matter hyperintensities with external capsule bilaterally (1, arrows), anterior temporal lobe, and pons involvement (2, arrows). (B) Pedigree of the CADASIL family. Filled symbols represent affected individuals. Dot within symbol represents asymptomatic carrier. Arrow indicates the proband. Plus signs indicate subject carriers of the mutation; dashes indicate indicate noncarriers. (C) Schematic representation of NOTCH3 intron 3 (and portions of exon 3 and 4) from wild-type and mutated sequence (c.34126_24delAAC). Putative BPS are indicated; the 3 nucleotides deleted are boxed. (D) Agarose gel electrophoresis of the products obtained after RT-PCR amplification with primers Afw and Crv. Lane 1: RT-PCR from subject IV-9; lane 2: RT-PCR from subject IV-8; lane 3: RT-PCR from subject III-5; lane 4: RT-PCR from control subject. M, molecular size markers. (E) Schematic representation of expected RT-PCReamplified cDNA. RT-PCR with primers Afw and Crv amplifies part of exon 2, exon 3 and 4 and part of exon 5. Correct splicing results in the excision of intron 3, giving a fragment of 639 bp. The unspliced product has a size of 714 bp and contains the complete 75 bp of intron 3 with the 3-base deletion.

the PCR product from the cDNA of the proband (III-5) and his son carrying the mutation (IV-8), compared with a single band from control cDNA (Fig. 1D). The sizes of the PCR products were those expected for a wild-type transcript (lower band), and 1 retaining intron 3 (upper band) (Fig. 1E). Direct cDNA sequencing confirmed an in-frame 75-bp insertion (corresponding to intron 3 with the AAC bases deleted), which was predicted to encode an extra 25 amino acids, including one cysteine, between codons 114 and 115d the largest amino acid insertion ever reported in mutant Notch3. 4. Discussion In this article, we report the characterization of a novel c.34126_24delAAC deletion mutation in intron 3 of the NOTCH3 gene, leading to aberrant splicing and associated with a pathologically confirmed CADASIL phenotype in an Italian family. In recent years, many splicing mutations have been reported to affect the splicing of pre-mRNA. Most of them involve donor or acceptor splice sites, although disease-causing mutations in the BPS are not frequently encountered (listed in Bishop et al., 2010). In this family, in silico analysis of intron 3 of the NOTCH3 gene detected 2 putative BPS, BPS1 and BPS2, and predicted that a new BPS (BPS3) would be formed following the c.341-26_24delAAC, with a lower score (Fig. 1C, and Supplementary material Table 1 and Fig. 1). Many findings indicate that a mutated BPS is easily

replaced by a new BPS, with only moderately reduced splicing efficiency (Reed and Maniatis, 1988); nevertheless, our study suggests that this is not always the case. The aberrant splicing found in our family suggests that the short length of the intron 3 (78 bp) might render it highly sensitive to any alteration within the BPS (Kralovicova et al., 2006). As an alternative, the 3-base deletion in intron 3 might decrease the intron length below the threshold value for splicing, which is 70 to 80 nucleotides for a vertebrate intron (Vansanten and Spritz, 1985). As reported previously (Auffray et al., 2006; Wang et al., 2002), deletions in small introns are deleterious for proper splicing because of the constraint on intron size. The causative relationship between c.341-26_24delAAC mutation in the NOTCH3 gene and CADASIL is strongly suggested by the following observations: (1) the mutation segregates with the disease phenotype in this family and is absent from healthy individuals; (2) there are no other NOTCH3 mutations that could account for the aberrant splicing observed; and (3) the unspliced intronic sequence contains the 3-bp deletions. Another point of interest concerns the late age at onset of clinical manifestations in this family, as all of the affected members presented the first symptom of disease, typically stroke and/or progressive cognitive deterioration, after the age of 60, in contrast to the usual temporal profile of clinical manifestation. This homogeneously late onset suggests a possible association with the

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particular intronic mutation described. With a few exceptions (Arboleda-Velasquez et al., 2002; Bianchi et al., 2010), the great majority of previous data shows no relationship between genotype and clinical phenotype. The finding of a mutation affecting splicing that is not easily identifiable on genomic DNA with exon flanking primers suggests that particular attention should be given to full mutational analysis of small introns. Because no NOTCH3 mutation is identifiable in 5% to 10% of GOM-positive CADASIL patients (Joutel et al., 1997; Peters et al., 2005), our findings suggest that NOTCH3 mutations in the genetically undefined cases may be located deep in the introns; however, the possibility that large deletion or duplication mutations or other genes could be involved cannot be ruled out. Thanks to the latest sequence analysis techniques, a far greater number of sequence variations will be detected in the near future, especially in introns. This will entail the need to determine their clinical significance. For this reason, assays to evaluate splicing will become more necessary, including in the diagnostic setting of CADASIL. Disclosure statement There are no conflicts of interest for any authors. Acknowledgements The authors thank the patients and their family members for their cooperation in the study. We are grateful to Ermelinda Tarquini for the fibroblast cultures; we also thank Leonardo Pantoni for his helpful comments on the manuscript. Preliminary data of this study have been presented as an oral communication at the 40th Congress of the Italian Neurological Society in Padua, Italy, November 21e25, 2009. Funding/Support. This work was supported in part by grants from the Fondazione Monte dei Paschi (N 38888) and MIUR (prot. 20095JPSNA_005) (Dotti), and by the Regione Toscana (Regional Health Research Program 2009) (Federico). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2013.03.005.

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