Absence of mtDNA mutations in leukocytes of CADASIL patients
© Abu-Amero et al; licensee BioMed Central Ltd. 2008
Received: 01 May 2008
Accepted: 30 May 2008
Published: 30 May 2008
Ultrastructural and biochemical abnormalities of mitochondria have been reported in skeletal muscle biopsies of CADASIL patients with mutations in the NOTCH3 nuclear gene. Additionally, it was proposed that NOTCH3 gene mutations may predispose the mitochondrial DNA (mtDNA) to mutations.
We sequenced the entire mitochondrial genome in five Arab patients affected by CADASIL.
The mean number of mtDNA sequence variants (synonymous and nonsynonymous) in CADASIL patients was not statistically significantly different from that in controls (p = 0.378). After excluding haplogroup specific single nucleotide polymorphisms (SNPs) and proved silent polymorphisms, no known or novel pathologic mtDNA mutation(s) could be detected in any patient. Additionally, there was no difference in the prevalence of different mitochondrial haplogroups between patients and controls.
Our study group is too small for any valid conclusion to be made. However, if our observation is confirmed in larger study group, then mtDNA mutations or mitochondrial haplogroups may not be important in the pathogenesis of CADASIL.
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (MIM 125310) is characterized by degeneration of vascular smooth muscle cells (VSMC) of nearly all tissues studied to date. The disease is caused by mutations of the NOTCH3 gene encoding the transmembrane receptor NOTCH3, which is expressed predominantly in VSMC . Electron microscopic examination of muscle and nerve biopsies from CADASIL patients with NOTCH3 mutations revealed enlarged mitochondria with needle-like calcium precipitates . A muscle biopsy and brain magnetic resonance spectroscopic imaging, for a CADASIL patient with frameshift deletion in the NOTCH3 gene, showed signs of mitochondrial impairment (ultrastructural mitochondrial abnormalities and increased parenchymal brain lactate respectively) . Ultrastructural examination of Fibroblasts and myobalsts from a CADASIL patient revealed mitochondrial aberration and reduced numbers. The patient harbors a NOTCH3 mutation and a mitochondrial mutation (5650 G> A) in the tRNA (Ala) gene . Finnila et al. assumed that the co-occurrence of the two mutations is not coincidental and that mutations in the NOTCH3 gene may predispose the mtDNA to mutations . Sequencing the entire mtDNA coding region in 77 CADASIL patients and 192 matching controls revealed the presence of higher number of polymorphisms among patients in comparison to controls . A significant decrease in the activity of complex I was noted in a muscle biopsy taken from a Spanish CADSAIL patient and histochemical analysis of his affected family members showed the presence of ragged-red fibers with abnormal cytochrome c oxidase staining . Dotti and others looked for common pathogenic mtDNA mutations in DNA extracted from the skeletal muscles of one CADASIL patient, and detected none . Muscle biopsies were not available to us, but given that mitochondrial dysfunctions can now be detected in circulating leukocytes of patients with various mitochondrial disorders [7–10], we sequenced the full mitochondrial genome from DNA extracted from circulating leukocytes of five CADASIL patients in order to search for any pathogenic mtDNA mutation.
Control subjects included: 1) 159 Saudi Arabian blood donors at the King Faisal Specialist Hospital and Research Centre who represented the spectrum of the Saudi population; 2) 50 healthy Sudanese individuals; 3) 57 Egyptians; 4) 108 healthy individuals from Yemen and 5) 120 individuals from gulf countries (Oman, Qatar, Kuwait and Bahrain). All Arab subjects in our repository reported no symptomatic metabolic, genetic, ocular or neurological disorders on an extensive questionnaire regarding family history, past medical problems and current health.
Sample collection and DNA extraction
Ten ml of peripheral blood were collected in EDTA tubes from all participating individuals after obtaining their written informed consent approved by the KFSH-IRB. DNA was extracted from whole blood samples of all CADASIL patients and controls using the PUREGENE DNA isolation kit from Gentra Systems (Minneapolis, USA).
mtDNA amplification and sequencing
The entire mitochondrial genome was amplified in all patients and controls using primers and amplification conditions described previously . Each successfully amplified fragment was directly sequenced using the same primers used for amplification and the BigDye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). Samples were run on the ABI prism 3100 sequencer (Applied Biosystems).
Sequence analysis of the mitochondrial DNA genome
The full mitochondrial genome was sequenced. Sequencing results were compared to the corrected Cambridge reference sequence . All fragments were sequenced in both forward and reverse directions at least twice for confirmation of any detected variant. All sequence variants from both CADASIL patients and controls were compared to the Mitomap database , the Human Mitochondrial Genome Database , GenBank , and Medline listed publications. Reported homoplasmic synonymous or non-synonymous (NS; resulting in amino acid change) polymorphisms used predominantly for haplogroup analysis were excluded from further consideration .
Prediction of pathogenicity
Pathologic characteristics of each NS sequence change, in both CADASIL patients and controls, were estimated according to a combination of standard pathogenicity criteria  and an evaluation of interspecies conservation using the PolyPhen database . Therefore, a NS nucleotide change was considered potentially pathologic, if it met all of the following criteria, when applicable: 1) it was not found in at least 100 controls of matching ethnicity; 2) it changed a moderately or highly conserved amino acid; 3) it was assessed as possibly or probably pathologic by PolyPhen; 4) it was not established as one of the haplogroup-specific SNPs and 5) It was not reported as an established polymorphism in MITOMAP database, the Human Mitochondrial Genome Database, GenBank, and Medline listed publications.
Clinical findings and NOTCH3 mutation detected in these CADASIL patients have been described previously 
Here, we sequenced the full mtDNA genome in five CADASIL patients and 159 ethnicity matching controls. The mean number of sequence changes, relative to the Cambridge reference sequence, detected in CADASIL patients (n = 5) was 18 SD 0.707 and among healthy controls (n = 159) was 17.4 SD 1.51. The difference between the two groups was not statistically significant (p = 0.378). All D-loop variants detected in CADASIL patients were also present in controls.
Non-synonymous mtDNA sequence changes detected in CADASIL patients
Amino Acid substitution
Base substitution type
TM domain of ND1 gene
TM domain of ND5 gene
TM domain of ND6 gene
Outside the functional domain of CYTB gene
Outside the functional domain of CYTB gene
Summary of the NOTCH3 mutations and mitochondrial abnormalities detected in CADASIL patients
NS+ mtDNA sequence changes
Mitochondrial haplogroup Of CADASIL patients
Percentage of mitochondrial haplogroup in controls (n = 552)
c.406 C> T
c.1568 C> T
c.406 C> T
c.475 C> T
We have previously described five Arab patients belonging to five unrelated pedigrees with a clinical phenotype typical of CADASIL. NOTCH3 mutations were found in four of those and we concluded that CADASIL occurs in Arabs, with clinical phenotype and genotype similar to that in other ethnic groups . As there has been considerable increase in the number of studies demonstrating mitochondrial abnormalities in CADASIL patients, we sequenced the entire mitochondrial genome with aim of detecting established or potentially pathogenic mtDNA sequence changes. One study has shown a higher number of mtDNA-polymorphisms among CADASIL patients in comparison to healthy controls and detected three pathogenic mtDNA mutations in three separate CADASIL pedigrees . However, this was not the case in our study as the number of polymorphisms was similar in both patients and controls. Additionally, we could not detect any pathogenic mtDNA mutation among our study group. The differences in mtDNA-polymorphisms observed in their study could be as a result of differences in haplogroup frequencies between CADASIL patients and controls as was stated . Only 3/77 (3.9%) of their CADASIL pedigrees had pathogenic mtDNA mutations and therefore, this finding cannot be taken as a complete picture. In light of our findings and others [4–6], previous hypothesis that NOTCH3 contribution to mtDNA maintenance and that, mutations in NOTCH3 increase the frequency of mutations in mtDNA  may not be compelling enough. Additionally, our CADSIL patients were assigned into their respective mitochondrial haplogroup. Each patient had a unique haplogroup and those were also present in Arab healthy population . We could not calculate the statistical significant differences in the mitochondrial haplogroup frequencies between controls and patients due to the fact that each patient had a unique haplogroup. Consequently, the number was too small to make a statistical comparison and as a result we could not reach a conclusion whether mitochondrial haplogroup is a risk factor for CADASIL or not. However, if our observation is confirmed in larger study group, then mtDNA mutations or mitochondrial haplogroups may not be important in the pathogenesis of CADASIL. This does not exclude the fact that other mitochondrial abnormalities, such as alteration in mitochondrial respiration or ultra-structure, could be involved.
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
Ethylene Diamine Tetra-acetic Acid
Magnetic resonance imaging
Transient ischemic attacks.
- Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, Alamowitch S, Domenga V, Cecillion M, Marechal E, et al: Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996, 383 (6602): 707-710. 10.1038/383707a0.View ArticlePubMedGoogle Scholar
- Schroder JM, Zuchner S, Dichgans M, Nagy Z, Molnar MJ: Peripheral nerve and skeletal muscle involvement in CADASIL. Acta neuropathologica. 2005, 110 (6): 587-599. 10.1007/s00401-005-1082-9.View ArticlePubMedGoogle Scholar
- Dotti MT, De Stefano N, Bianchi S, Malandrini A, Battisti C, Cardaioli E, Federico A: A novel NOTCH3 frameshift deletion and mitochondrial abnormalities in a patient with CADASIL. Archives of neurology. 2004, 61 (6): 942-945. 10.1001/archneur.61.6.942.View ArticlePubMedGoogle Scholar
- Finnila S, Tuisku S, Herva R, Majamaa K: A novel mitochondrial DNA mutation and a mutation in the Notch3 gene in a patient with myopathy and CADASIL. Journal of molecular medicine (Berlin, Germany). 2001, 79 (11): 641-647.View ArticleGoogle Scholar
- Annunen-Rasila J, Finnila S, Mykkanen K, Moilanen JS, Veijola J, Poyhonen M, Viitanen M, Kalimo H, Majamaa K: Mitochondrial DNA sequence variation and mutation rate in patients with CADASIL. Neurogenetics. 2006, 7 (3): 185-194. 10.1007/s10048-006-0049-x.View ArticlePubMedGoogle Scholar
- de la Pena P, Bornstein B, del Hoyo P, Fernandez-Moreno MA, Martin MA, Campos Y, Gomez-Escalonilla C, Molina JA, Cabello A, Arenas J, et al: Mitochondrial dysfunction associated with a mutation in the Notch3 gene in a CADASIL family. Neurology. 2001, 57 (7): 1235-1238.View ArticlePubMedGoogle Scholar
- Kunz D, Luley C, Winkler K, Lins H, Kunz WS: Flow cytometric detection of mitochondrial dysfunction in subpopulations of human mononuclear cells. Analytical biochemistry. 1997, 246 (2): 218-224. 10.1006/abio.1997.2007.View ArticlePubMedGoogle Scholar
- Marriage BJ, Clandinin MT, MacDonald IM, Glerum DM: The use of lymphocytes to screen for oxidative phosphorylation disorders. Analytical biochemistry. 2003, 313 (1): 137-144. 10.1016/S0003-2697(02)00539-0.View ArticlePubMedGoogle Scholar
- Zhang HX, Du GH, Zhang JT: Assay of mitochondrial functions by resazurin in vitro. Acta pharmacologica Sinica. 2004, 25 (3): 385-389.PubMedGoogle Scholar
- Abu-Amero KK, Bosley TM: Detection of mitochondrial respiratory dysfunction in circulating lymphocytes using resazurin. Arch Pathol Lab Med. 2005, 129 (10): 1295-1298.PubMedGoogle Scholar
- Maca-Meyer N, Gonzalez AM, Larruga JM, Flores C, Cabrera VM: Major genomic mitochondrial lineages delineate early human expansions. BMC genetics. 2001, 2: 13-10.1186/1471-2156-2-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N: Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet. 1999, 23 (2): 147-10.1038/13779.View ArticlePubMedGoogle Scholar
- Brandon MC, Lott M, Nguyen KC, Spolim S, Navathe SB, Baldi P, Wallace D: MITOMAP: A human mitochondrial genome database–2004 update. Nucleic Acids Res. 2005, D611-613. [http://www.mitomap.org]33 Database
- Ingman M, Gyllensten U: mtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences. Nucleic Acids Res. 2006, D749-751. 10.1093/nar/gkj010. 34 Database
- Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL: GenBank. Nucleic Acids Res. 2008, D25-30. 36 Database
- Bandelt HJ, Salas A, Bravi CM: What is a 'novel' mtDNA mutation–and does 'novelty' really matter?. Journal of human genetics. 2006, 51 (12): 1073-1082. 10.1007/s10038-006-0066-5.View ArticlePubMedGoogle Scholar
- Chinnery PF, Howell N, Andrews RM, Turnbull DM: Mitochondrial DNA analysis: polymorphisms and pathogenicity. Journal of medical genetics. 1999, 36 (7): 505-510.PubMed CentralPubMedGoogle Scholar
- Ramensky V, Bork P, Sunyaev S: Human non-synonymous SNPs: server and survey. Nucleic Acids Res. 2002, 30 (17): 3894-3900. 10.1093/nar/gkf493.PubMed CentralView ArticlePubMedGoogle Scholar
- Bohlega S, Al Shubili A, Edris A, Alreshaid A, Alkhairallah T, AlSous MW, Farah S, Abu-Amero KK: CADASIL in Arabs: clinical and genetic findings. BMC medical genetics. 2007, 8: 67-10.1186/1471-2350-8-67.PubMed CentralView ArticlePubMedGoogle Scholar
- Abu-Amero K, Larruga JM, Cabrera VM, Gonzalez AM: Mitochondrial DNA structure in the Arabian Peninsula. BMC Evol Biol. 2008, 8: 45-10.1186/1471-2148-8-45.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.