European Journal of Neurology 2008, 15: 1006–1008
doi:10.1111/j.1468-1331.2008.02222.x
SHORT COMMUNICATION
No evidence of association between frontotemporal dementia and major European mtDNA haplogroups G. Rosea, T. Longoa, R. Malettab, G. Passarinoa, A. C. Brunib and G. De Benedictisa a
Department of Cell Biology, University of Calabria, Rende, Italy; and bRegional Neurogenetic Centre, ASL 6, Lamezia Terme, CZ, Italy
Keywords:
frontotemporal dementia, mtDNA, risk factors Received 2 April 2008 Accepted 23 May 2008
Background and purpose: Mitochondrial DNA (mtDNA) inherited variability (haplogroup/sub-haplogroup) is currently emerging as not being neutral with respect to several complex traits like neurodegenerative diseases. Here we investigated the association of European mtDNA haplogroups/sub-haplogroups with frontotemporal dementia (FTD). Method and Results: A case-control study was carried out on 114 patients with FTD (68 sporadic and 46 familial) and 180 controls, matched for age, gender and ethnicity. No association was found. Conclusions: European mtDNA haplogroups/sub-haplogroups are unlikely to play a major role in the risk of developing the disease.
Introduction Frontotemporal dementia (FTD) is a complex and heterogeneous disease, more common than previously thought, whose aetiology still remains elusive. Although causative mutations have rarely been found in a few genes (such as MAPT and PRGN, accounting for up to 2.9 and 4.8% of the cases) [1], FTD usually appears as a complex trait where susceptibility is due to the common variability in a number of genes [2]. mtDNA variability may affect susceptibility to neurodegenerative diseases, because of the essential role played by mitochondria in energy metabolism and apoptosis. In fact, mitochondria are essential for neuronal function because neurons are highly dependent on oxidative phosphorylation for their energetic needs, and oxidative damage is observed in all classes of organic molecules (proteins, lipids, nucleic acids and sugars) that are critical for structural and functional neuronal integrity [3]. mtDNA variation has been well characterized at the population level. Based on the information obtained from RFLP analysis and from sequence analysis of the hypervariable segments HVS-I and HVS-II (control region), several haplogroups (clusters of ancient shared variants) have been described [4,5]. Whether mtDNA-inherited variability does contribute to FTD has not been investigated till now. Aim of this work was to test the hypothesis that the major European mtDNA haplogroups play a role in the onset of FTD. Correspondence: Prof. Giuseppina Rose, Department of Cell Biology, University of Calabria, 87036 Rende, Italy (tel.: +39 984 492931; fax: +39 984 492911; e-mail:
[email protected]).
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Materials and methods Sample
A sample of 114 unrelated patients with FTD was recruited at the Regional Neurogenetics Centre (Calabria, southern Italy). Patients without other affected members in the family were classified as affected by sporadic FTD (n = 68 subjects). On the contrary, patients with a positive family history of FTD were classified as affected by familial FTD (n = 46 subjects). Diagnosis of FTD was assessed by using multiple operational criteria and was based on specific clinicalneuropsychological features and neuroradiological profiles (for details see ref. [2] and references therein). A control group of 180 unrelated subjects matched for age, sex and ethnicity was recruited in the same population. The same complete set of clinical-laboratory procedures and neurological assessment of cognitive status used for patients were also performed in the control group. In addition, a previous screening carried out by using sequence analysis on MAPT gene failed to find any mutations in this sample [2]. The age range in each of the three sample groups was as follows: 40–80 years in the sporadic FTD group (median age 68.0 years, 30 men and 38 women); 45–80 years in the familial FTD group (median age 54.5 years; 23 men and 23 women) and 40–80 years in the control group (median age 66.5 years, 84 men and 96 women). The study was approved by the Ethical Committee of the University of Calabria and informed consent for research purposes was obtained from all individuals involved in the study; for disabled patients with FTD consent was given by their legal tutors. 2008 The Author(s) Journal compilation 2008 EFNS
FTD and major European mtDNA haplogroups
Analysis of mtDNA variation
All the PCR amplifications were carried out in a 25-ll reaction mixture containing 200 ng of genomic DNA, 1 U Taq DNA polymerase (Eppendorf AG, Hamburg, Germany), 0.3 lM of each primer, 0.2 mM dNTPs, 1.5 mM Mg(OAc)2 and 1· reaction buffer. Amplifications were performed in an Eppendorf thermal cycler. Cycling conditions were the same for all the fragments, but with different specific annealing temperatures. Specifically, the initial denaturation at 93C for 30 s was followed by 35 cycles at 93C for 15 s, specific primer annealing temperature for 20 s, 72C for 1 min and a final extension of 72C for 12 min. Sequences of each pair of primers and annealing temperatures used in PCR are listed in Table S1. For haplogroup typing, the amplified fragments were digested with the appropriate enzyme (Table S2) and separated by using 2% agarose gel electrophoresis. By using this procedure, each mtDNA was assigned to one of the nine haplogroups (H, I, J, K, T, U, V, W and X) specific to Europeans [4]. HVS-I, HVS-II and coding region diagnostic markers for sub-haplogroup typing were checked by using sequence analyses (Table S3). The amplified fragments were purified by using QIAquick PCR purification Kit (Qiagen, Hilden, Germany) and sequenced by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer, Waltham, MA, USA) on an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA, USA). Cycle sequencing reaction mixtures contained 4 ll of terminator ready reaction mix, 200 ng of template, 3.2 pmol of each primer, 4 ll of 5· reaction buffer in a total volume of 20 ll. Sequencing was performed for 25 cycles at 96C for 10 s, 50C for 5 s, 60C for 4 min in an Eppendorf thermal cycler. The
Table 1 mtDNA haplogroup frequency distributions in FTD patients and controls
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extension products were purified by using Amersham spin columns (Amersham Biosciences, Piscataway, NJ, USA). Sequences were aligned by using GENALYS 2.0 beta software and compared with the revised Cambridge reference sequence (rCRS). Statistical analysis
The null hypothesis of homogeneity between haplogroup/sub-haplogroup frequency distributions in the pair of samples was tested by using permutation tests [6]. The tests were performed on MATLAB 6.1 by using 10 000 random shuffles of haplotypes between the relevant groups.
Results Patients with frontotemporal dementia and healthy controls were screened for mtDNA haplogroups (see Table 1) and sub-haplogroups (Table S4). No significant difference was found between patients with sporadic FTD and controls or between patients with familial FTD and controls either in the frequency distribution of haplogroups (P = 0.08 and P = 0.51) or sub-haplogroups (P = 0.97 in both cases). The study was further extended by sequencing the HVS-I region. Table S5 reports all mtDNA sequence variants found in the HVS-I region, with haplotype absolute frequencies in each sample. Again, no difference was found between patients with sporadic or familial FTD and controls (P = 0.98 in both cases).
Discussion A fundamental requirement for the reliability of association studies is the comparison of rigorously
Patients with sporadic FTD
Patients with familial FTD
Controls
mtDNA haplogroups
N
% ± SE
N
% ± SE
H I J K T U V W X Others Total
30 1 4 10 5 7 3 1 4 3 68
44.1 1.5 5.9 14.7 7.4 10.3 4.4 1.5 5.9 4.4 100
16 2 3 4 7 8 1 1 0 4 46
34.8 4.3 6.5 8.7 15.2 17.4 2.2 2.2 0.0 8.7 100
± ± ± ± ± ± ± ± ± ±
6.0 1.5 2.9 4.3 3.2 3.7 2.5 1.5 2.9 2.5
± ± ± ± ± ± ± ± ± ±
7.0 3.0 3.6 4.2 5.3 5.6 2.2 2.2 0.0 4.2
N
% ± SE
49 8 24 22 21 18 2 4 9 23 180
27.2 4.4 13.3 12.2 11.7 10.0 1.1 2.2 5.0 12.8 100
± ± ± ± ± ± ± ± ± ±
3.3 1.5 2.5 2.4 2.4 2.2 0.8 1.1 1.6 2.5
Absolute (N) and relative (%) frequencies with standard errors (±SE) are shown. mtDNAs not classifiable within any specific haplogroup were grouped as others [4].
2008 The Author(s) Journal compilation 2008 EFNS European Journal of Neurology 15, 1006–1008
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phenotyped cases and well-matched controls [7]. To this purpose, we enrolled only patients and controls of clear Calabrian origin, matched for ethnicity, genetic origin, sex and age. Moreover, a team of neurologists, psychologists and physicians carried out an exhaustive diagnosis of FTD. Our analysis failed to reveal an association between mtDNA-inherited variability and FTD, even though the analysis has been extended up to the sub-haplogroup level. This negative finding can be explained by two alternative hypotheses: (i) major European mtDNA haplogroups do not affect susceptibility to FTD, at least in our population; (ii) the sample size, although is one of the largest ones including patients with FTD, has no sufficient statistical power to reveal these associations either because the influence of mtDNA haplogroup on FTD is quite low or because only some rare mtDNA haplotypes contribute to FTD [8]. On the other hand, when the disease is quite rare, the collection of samples having both phenotypic homogeneity and adequate size is challenging. The problem of an adequate sample size greatly increases when sub-haplogroup or sequence analyses are performed, and it is still an open question at what level the analysis of mtDNA variability should be stopped to reveal phenotype–genotype associations. In any case, although negative, the results presented here may provide a reliable starting point for further investigations on the relationships between FTD and the mitochondrial genome.
Table S4. Mitochondrial DNA (mtDNA) sub-haplogroup (Sub-Hg) frequency distributions in patients with FTD and controls. Absolute (N) and relative (%) frequencies with standard errors (±SE) are shown. The asterisk denotes mtDNAs that were not classifiable within specific sub-haplogroups. Table S5. Variant sites found in mitochondrial DNA HVS-I. For each haplotype, the absolute frequency in patients with sporadic frontotemporal dementia (FTD) (S), familial FTD (F) and controls (C) is reported. rCRS refers to revised Cambridge reference sequence (MITOMAP: http://www.mitomap.org, 2007). The position of the mutations is given according to rCRS less 16 000. A total of 174 haplotypes were found, 24 of which were exclusive of familial FTD, 27 of sporadic FTD and 99 were exclusive of controls; the remaining eight haplotypes were shared between patients with FTD and controls. The material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j.14681331.2008.02222.x (This link will take you to the article abstract). Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
References Acknowledgements This study was supported by the Regional Health Department – Calabria Region, by the finalized project of the Italian Health Ministry (ÔLa Calabria come isola geneticaÕ No. 42 of 26 November 2003, approved on 11 October 2004) and by the Italian Ministry of University and Research (PRIN, 2004; to GP).
Supplementary material The following supplementary material is available for this article online: Table S1. PCR primers and conditions used to define haplogroups (Hg) and *sub-haplogroups (Sub-Hg), and for PCR amplification of HVS-I and HVS-II regions (see ref. [4,5]). Table S2. Diagnostic restriction sites defining mtDNA haplogroups (Hg). Table S3. Mitochondrial DNA diagnostic markers in HVS-I, HVS-II and in the coding region by which subhaplogroups (Sub-Hg) are defined (MITOMAP: http:// www.mitomap.org).
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2008 The Author(s) Journal compilation 2008 EFNS European Journal of Neurology 15, 1006–1008
