Absence of mtDNA mutations in leukocytes of CADASIL patients

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 [1]. Electron microscopic examination of muscle and nerve biopsies from CADASIL patients with NOTCH3 mutations revealed enlarged mitochondria with needle-like calcium precipitates [2]. 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) [3]. 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 [4]. 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 [4]. 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 [5]. 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 [6]. Dotti and others looked for common pathogenic mtDNA mutations in DNA extracted from the skeletal muscles of one CADASIL patient, and detected none [3]. 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.

Clinical findings and NOTCH3 mutation detected in these CADASIL patients have been described previously [19]

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.

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 [19]. 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 [5]. 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 [5]. 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 [4] 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 [20]. 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.