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Mitochondrial DNA, Early Online: 1–6! 2013 Informa UK Ltd. DOI: 10.3109/19401736.2013.836508
ORIGINAL ARTICLE
Association study of mitochondrial DNA polymorphisms with type 2diabetes in Tunisian population
Sana Hsouna1,2*, Nizar Ben Halim1*, Khaled Lasram1, Imen Arfa1, Henda Jamoussi1,3, Sonia Bahri4,Slim Ben Ammar4, Najoua Miladi2, Abdelmajid Abid1,3, Sonia Abdelhak1, and Rym Kefi1
1Biomedical Genomics and Oncogenetics Laboratory (LR 11 IPT 05), Institut Pasteur de Tunis, Universite El Manar de Tunis, Tunis, Tunisia,2Child Neurological Diseases Unit (05/UR/08-02), Faculte de Medecine de Tunis, Tunis, Tunisia, 3Service de consultation externe et exploration
fonsctionnelle, Institut National de Nutrition, Tunis, Tunisia, and 4Department of Biochemistry, Institut Pasteur de Tunis, Tunis, Tunisia
Abstract
Mitochondrial DNA (mtDNA) variation may play an important role in the pathogenesis of type 2diabetes (T2Ds). In this study, we aimed to explore whether mtDNA variants contribute to thesusceptibility to T2Ds in a Tunisian population. The hypervariable region 1 (HVS1) of the mtDNAof 64 T2Ds patients and 77 healthy controls was amplified and sequenced. Statistical analysiswas performed using the STATA program. Analysis of the total screened variants (N¼ 88) fromthe HVS1 region showed no significant difference in the distribution of all polymorphismsbetween T2Ds and controls, except for the variant G16390A which was more frequent in T2Ds(15.9%) than in controls (5.4%) (p¼ 0.04). The association of G16390A was not detectedafter multivariate regression analysis. Similarly, analysis of the distribution of mitochondrialhaplogroups within our dataset showed 18 distinct major haplogroups with no significantdifference between T2Ds and controls. Except, the weakly association found for the G16390Avariant, our results showed that none of the tested polymorphisms from the HVS1 region havea major role in T2Ds pathogenesis in the studied Tunisian population even when taking intoaccount the population stratification.
Keywords
Hypervariable region 1, mitochondrial DNA,North Africa, Tunisia, type 2 diabetes
History
Received 2 March 2013Revised 30 July 2013Accepted 16 August 2013Published online 8 October 2013
Introduction
Type 2 diabetes (T2Ds) is a complex disease characterized byinsulin resistance in peripheral tissues with impaired insulinsecretion from pancreatic b cells (Tang et al., 2006). There isfurther evidence that oxidative stress plays a significant role inthe progression of b cell deterioration during the developmentof T2Ds (Sakuraba et al., 2002). In the Tunisian population, theprevalence of T2Ds reaches 9% of adults (Bouguerra et al., 2007;Ezzidi et al., 2009; Mehri et al., 2010), which is much highercompared with European populations but less frequent than thatobserved in Indian and some Asian populations (Lowell &Shulman, 2005). Previous studies have assessed the familialclustering as well as the matrilineal inheritance of T2Ds inTunisian patients (Arfa et al., 2007; Bouhaha et al., 2010).Similarly, other epidemiologic studies performed in Mauritania(Meiloud et al., 2013), Morocco (Benrahma et al., 2011), UnitedKingdom and Japan (Fuku et al., 2007; Thomas et al., 1994)revealed a Matrilineal inheritance of T2Ds.
Mitochondrial genome might contribute to the genetic etiologyof T2Ds and related metabolic disorders (Atig et al., 2009). Indeed,one particular form of diabetes is the mitochondrial inheriteddiabetes and deafness (MIDD) characterized by a matrilineal
inheritance associated with bilateral hearing defect in most of theaffected patients (Cormio et al., 2009). The most common mutationlinked to mitochondrial diabetes is the A3243G in the mitochon-drial tRNALeu gene. This mutation causes reduced binding of theamino acid to its transfer RNA leading to decreased synthesis ofproteins necessary for oxidative phosphorylation and reducedglucose-stimulated insulin secretion (Suzuki et al., 1997). InTunisia, only two studies have been performed to analyze theassociation of the A3243G mutation in the mitochondrial tRNALeu
gene with the MIDD and diabetes in Tunisian population (Bouhahaet al., 2010; Mkaouar-Rebai et al., 2007). Results showed a lowfrequency (1.07%) (Bouhaha et al., 2010) or absence of the A3243Gmutation in Tunisians with diabetes (Mkaouar-Rebai et al., 2007).
Different mitochondrial variants have been proposed to beassociated with diabetes such as the homoplasmic T3394C andG4491A in NADH dehydrogenase subunit I (ND1) and ND2genes, respectively (Guo et al., 2005; Liu et al., 2007; Poultonet al., 2002). Other polymorphisms such as T16189C andT16519C variants in the HVS1 were also closely associatedwith the susceptibility to T2Ds in Italian and Chinese populations(Liao et al., 2008; Navaglia et al., 2006). Nonetheless, theimplication of certain mitochondrial variants in T2Ds is stillcontroversial. That is the case for T16189C variant among others(Chinnery et al., 2005; Mohlke et al., 2005; Saxena et al., 2006).
In the Tunisian population, no studies have investigated theimpact of the HVS1 polymorphisms on T2Ds. Taking intoaccount the familial clustering and the maternal inheritance ofT2Ds in Tunisian patients, we proposed to study the implicationof the HVS1 region in the pathogenesis of T2Ds in the Tunisianpopulation.
*Both authors contributed equally to the present work.
Correspondence: Dr. Rym Kefi, Assistant Professor, PhD, BiomedicalGenomics and Oncogenetics Laboratory (LR 11 IPT 05), Institut Pasteurde Tunis, Universite El Manar de Tunis, Tunis, Tunisia. Tel: +21671843755. Fax: +216 71791833. E-mail: [email protected];[email protected]
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Methods
Subjects
Blood samples were collected from 141 unrelated Tunisiansubjects among whom 64 patients with T2Ds and 77 healthycontrols. T2Ds patients were diagnosed according to the WorldHealth Organization criteria (Alberti & Zimmet, 1998) andrecruited from National Institute of Nutrition of Tunis which isthe referral diabetes Medical Center in Tunisia. Control subjectswere chosen older than 40 years without first degree familyhistory of diabetes. Their glucose levels were normal (56.1 mmol/l), as described significantly related to diabetes in Tunisianpopulation (Bouguerra et al., 2007). Both patients and controls arerepresentative of various geographical regions of Tunisia.
Informed consent was obtained from all subjects. Clinicaldescription of subjects included gender, age, BMI (body massindex defined by ratio of weight in kilograms to square of heightin meters), blood glucose, total cholesterol, triglycerides andHDL cholesterol is shown in Table 1.
Genetic analysis
Genomic DNA was extracted from patient and control bloodsamples and the HVS1 was amplified and sequenced usingprocedures described by Stevanovitch et al. (2004). DNAsequencing was performed on ABI 3130 (Applied BiosystemsLife Technologies SAS, Saint Aubin, France). Sequences obtainedwere aligned and compared to the revised Cambridge ReferenceSequence (rCRS) using Blast 2 sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=blast2seq)and Seqscape software (V2.5) (Applied Biosystems, LifeTechnologies SAS, Saint Aubin, France).
Statistical analysis
Quantitative clinical data were presented as mean� SD (standarddeviation) and compared between cases and controls using theunpaired Student’s t test as well as the non-parametric Mann–Whitney test, used as needed to confirm significance. Categoricaldata were presented as numbers and percentages. The differencein the allele frequency of each variant was initially assessed by the�2-test. The strength of the association of variants with T2Ds wasestimated with the odds ratios (OR) with 95% confidenceintervals (CI). A logistic regression, adjusted for sex, age andBMI, was used in order to confirm the association. The differencein haplogroup distribution between T2Ds and control groupswas statistically tested through Fisher’s exact probability test.A p value less than 0.05 was considered as the level ofstatistical significance. Then, the power was computed for eachSNP (https://www.dssresearch.com/KnowledgeCenter/toolkitcalculators/statisticalpowercalculators.aspx), the overall powerwas calculated as the average power over all the studiedSNPs. At a¼ 0.05, this sample size provided 34.25% power
in detecting the T2Ds susceptibility variants. All the statisticalanalyses were performed with STATA statistical package (ver-sion 11.0) (StataCorp., College Station, TX).
Results
The basic clinical characteristics of the 141 Tunisian subjects areshown in Table 1. Triglyceride as well as blood glucose levelswere significantly higher in T2Ds patients than in controls(p50.05), whereas, no significant difference in the level of theHDL cholesterol was observed (p40.05). Similarly, no signifi-cant differences were observed between patients and controls forsex, BMI and total cholesterol. Patients were significantly olderthan controls (p50.05). This difference reflects the enrollmentcriteria for T2Ds (�40 years).
The analysis of the HVS1 sequences (from 16069np to16400np) in the 141 Tunisian subjects highlighted the presenceof 88 SNPs. According to the classification undertaken inprevious studies such as Wang et al.’s (2009), the 88 SNPscould be clustered into two groups: common variants and rarevariants. The first group defined as ‘‘common variants’’ wasrepresented by SNPs with Minor Allele Frequencies(MAF)410%. While the second group of ‘‘rare variants’’contains SNPs with MAF� 10. The common variants encom-passed C16223T, C16278T and C16294 which are observedwith higher frequencies in T2Ds patients than in controls andT16126C, T16189C, T16311C, which are found at higherfrequencies in controls than in T2Ds. In this group, theT16189C and C16223T variants showed the highest frequencies(430%) in both T2Ds patients and controls (Table 2). Theremaining 82 SNPs were grouped as ‘‘rare variants’’ (�10).
The 88 SNPs were statistically analyzed for associations withT2Ds. Most of them (87 SNPs) showed no significant difference inallele frequency distribution between patients and controls (Table2). In particular, we excluded the implication of the most commonT16189C variant, known to be related with T2Ds. Only one SNP:G16390A was found at a significantly higher frequency (10/63;15.9%) in T2Ds patients when compared to controls (4/74; 5.4%)(p¼ 0.04). This association of the G16390A found under aunivariate model, was not detected after multivariate logistic-regression with adjustment for sex, age and BMI (p¼ 0.11)(Table 2). Given that different mitochondrial variants affect healthand disease differentially depending on their mitochondrial gen-omic background (haplogroup) (Ji et al., 2012), we analyzed thedistribution of mitochondrial haplogroups within our dataset (Table3) and adjusted the relative risk associated to each SNP accord-ingly. Data showed 18 distinct major haplogroups in the overallsample with no significant difference on maternal lineagesdistribution between cases and controls (p¼ 0.97). Multivariatelogistic regression analysis stratified for mitochondrial hap-logroups showed that none of the screened mitochondrial vari-ants were significantly associated with susceptibility to T2Ds(Table 2).
Discussion
Previous studies have suggested that several mtDNA variants wereinvolved in the pathogenesis of T2Ds. The majority of thesestudies were performed on European and Asian populations(Bhat et al., 2007a, b; Liao et al., 2008; Liou et al., 2007; Mohlkeet al., 2005; Ruiz-Pesini et al., 2004; Salles et al., 2007; Tawataet al., 2000).
In this paper, we investigated the association of 88 SNPslocated in the HVS1 mtDNA region from 16069np to 16400npwith T2Ds in the Tunisian population. To our knowledge, thisis the first study of HVS1 mtDNA variants in a North Africanpopulation.
Table 1. Clinical description of Tunisian patients with T2Ds and controls.
Variable T2Ds (n¼ 64) Controls (n¼ 77) p Value
Gender (Men/Women) 31/28 45/31 0.44yAge (years) 60.5� 10.65 52.7� 12.14 2� 10�4z*BMI (kg/m2) 28.9� 4.60 27.7� 5.35 0.21zBlood glucose (mM) 11.7� 5.16 5.5� 0.41 10�4#*Total cholesterol (mM) 5.07� 128 4.8� 0.77 0.62#Triglycerides (mM) 1.8� 1.02 1.3� 0.72 0.02#HDL cholesterol (mM) 1.2� 0.40 1.4� 0.47 0.05#
Values are given as means� SD; *Significant p value (p50.05);yPearson’s chi square test; zStudent t test; #Mann–Whitney test.
2 S. Hsouna et al. Mitochondrial DNA, Early Online: 1–6
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Tab
le2
.A
llel
efr
equ
enci
eso
fm
tDN
Ap
oly
mo
rph
ism
sin
T2
Ds
pat
ien
tsan
dco
ntr
ols
.
Po
lym
orp
his
ms
T2
Ds
(%)
Co
ntr
ols
(%)
OR
(95
%C
I)p
Val
ue
ORy
(95
%C
I)p
Val
ue
ORz
(95
%C
I)p
Val
ue
Co
mm
on
po
lym
orp
his
ms
T1
61
26
C1
1/5
9(1
8.6
4)
16
/65
(24
.62
)0
.74
(0.3
1–
1.7
7)
0.5
00
.46
(0.1
6–
1.3
0)
0.1
40
.84
(0.3
4–
2.0
8)
0.7
1T
16
18
9C
19
/64
(29
.68
)2
4/7
7(3
1.1
6)
0.9
3(0
.45
–1
.92
)0
.85
0.8
3(0
.34
–1
.98
)0
.67
0.9
2(0
.44
–1
.90
)0
.82
C1
62
23
T2
8/6
4(4
3.7
5)
26
/77
(33
.76
)1
.52
(0.7
6–
3.0
4)
0.2
31
.27
(0.5
5–
2.9
6)
0.5
71
.68
(0.8
3–
3.4
0)
0.1
5C
16
27
8T
19
/64
(29
.68
)1
7/7
7(2
2.0
7)
1.4
9(0
.69
–3
.20
)0
.30
1.6
4(0
.62
–4
.30
)0
.32
1.4
7(0
.68
–3
.16
)0
.32
C1
62
94
T1
7/6
4(2
6.5
6)
12
/76
(15
.79
)1
.93
(0.8
3–
4.4
7)
0.1
21
.12
(0.4
2–
2.9
6)
0.8
21
.76
(0.7
5–
4.1
2)
0.1
9T
16
31
1C
17
/64
(26
.56
)2
0/7
4(2
7.0
3)
0.9
8(0
.46
–2
.08
)0
.95
0.9
5(0
.39
–2
.32
)0
.91
1.0
7(0
.49
–2
.32
)0
.86
Ra
rep
oly
mo
rph
ism
sC
16
06
9T
2/5
4(3
.70
)3
/62
(4.8
4)
0.7
6(0
.12
–4
.74
)0
.76
––
0.8
2(0
.13
–5
.12
)0
.83
C1
60
86
T1
/54
(1.8
5)
––
––
–0
.95
(0.9
0–
1.0
1)
0.0
9T
16
09
3C
4/5
7(7
.02
)3
/65
(4.6
2)
1.5
6(0
.33
–7
.34
)0
.57
5.3
0(0
.55
–5
1.3
5)
0.1
51
.64
(0.3
4–
7.8
1)
0.5
3C
16
11
1T
1/5
7(1
.75
)1
/65
(1.5
4)
1.1
4(0
.07
–1
8.9
2)
0.9
21
.44
(0.0
8–
26
.09
)0
.81
C1
61
14
A3
/57
(5.2
6)
––
0.0
6–
–1
.33
(0.0
8–
22
.89
)0
.84
T1
61
24
C2
/59
(3.3
9)
3/6
5(4
.62
)0
.72
(0.1
1–
4.5
3)
0.7
30
.82
(0.1
0–
6.7
6)
0.8
50
.68
(0.1
1–
4.2
6)
0.6
8G
16
12
9A
6/5
9(1
0.1
7)
5/6
5(7
.69
)1
.43
(0.4
1–
4.9
7)
0.5
82
.21
(0.3
8–
12
.64
)0
.37
1.6
2(0
.45
–5
.79
)0
.46
G1
61
45
A1
/59
(1.6
9)
3/6
5(3
.08
)0
.37
(0.0
4–
3.7
5)
0.3
80
.49
(0.0
3–
6.7
6)
0.6
00
.54
(0.0
5–
6.2
4)
0.6
2C
16
14
7T
1/6
0(1
.67
)–
–0
.29
––
––
C1
61
48
T1
/60
(1.6
7)
2/6
5(3
.08
)0
.56
(0.0
5–
6.4
0)
0.6
30
.48
(0.0
4–
5.8
6)
0.5
70
.66
(0.0
6–
7.5
7)
0.7
4G
16
15
3A
–1
/68
(1.4
7)
–0
.35
––
––
A1
61
62
G2
/63
(3.1
7)
––
0.1
2–
–0
.97
(0.9
2–
1.0
2)
0.2
7A
16
16
3G
4/6
3(6
.35
)5
/75
(6.6
7)
0.9
5(0
.24
–3
.71
)0
.94
1.0
5(0
.24
–4
.63
)0
.95
0.7
5(0
.19
–3
.06
)0
.69
C1
61
68
T1
/63
(1.5
9)
3/7
6(3
.95
)0
.39
(0.0
4–
3.9
2)
0.4
10
.40
(0.0
4–
4.2
2)
0.4
50
.50
(0.0
5–
5.0
4)
0.5
5C
16
16
9T
2/6
3(3
.17
)1
/76
(1.3
2)
2.4
6(0
.21
–2
8.1
5)
0.4
52
.24
(0.1
9–
26
.42
)0
.52
4.1
5(0
.34
–5
0.8
4)
0.2
7T
16
17
2C
3/6
3(4
.76
)7
/76
(9.2
1)
0.4
9(0
.12
–2
.01
)0
.31
0.6
7(0
.15
–3
.00
)0
.60
0.5
2(0
.13
–2
.10
)0
.36
A1
61
83
C1
/64
(1.5
6)
4/7
7(5
.19
)0
.29
(0.0
3–
2.7
1)
0.2
50
.27
(0.0
2–
3.0
3)
0.3
40
.28
(0.0
3–
2.6
1)
0.2
71
61
83
del
A2
/64
(3.1
2)
––
0.1
2–
––
–C
16
18
4T
1/6
4(1
.56
)–
–0
.27
––
0.9
6(0
.92
–1
.02
)0
.18
C1
61
85
T–
3/7
7(3
.89
)–
0.1
1–
–0
.96
(0.9
1–
1.0
2)
0.1
7C
16
18
6T
4/6
4(6
.25
)5
/77
(6.4
9)
0.9
6(0
.24
–3
.75
)0
.95
1.3
8(0
.28
–6
.86
)0
.69
0.8
1(0
.20
–3
.24
)0
.77
C1
61
87
T6
/64
(9.3
7)
5/7
7(6
.49
)1
.49
(0.4
3–
5.1
6)
0.5
31
.22
(0.3
3–
4.5
0)
0.7
71
.63
(0.4
7–
5.6
8)
0.4
5C
16
18
8T
1/6
4(1
.56
)1
/77
(1.2
9)
1.2
1(0
.07
–1
9.8
8)
0.8
9–
–1
.17
(0.0
7–
19
.15
)0
.91
C1
61
88
G1
/64
(1.5
6)
2/7
7(2
.59
)0
.59
(0.0
5–
6.7
9)
0.6
70
.52
(0.0
4–
6.2
5)
0.6
10
.72
(0.0
6–
8.3
0)
0.8
0T
16
18
9A
2/6
4(3
.12
)–
–0
.12
––
0.9
6(0
.91
–1
.02
)0
.17
16
18
9d
elT
1/6
4(1
.56
)1
/77
(1.2
9)
1.2
1(0
07
–1
9.8
8)
0.8
9–
–1
.17
(0.0
7–
19
.30
)0
.91
C1
61
92
T5
/64
(7.8
1)
3/7
7(3
.89
)2
.09
(0.4
7–
9.2
0)
0.3
21
.62
(0.2
6–
9.9
6)
0.6
01
.95
(0.4
4–
8.5
7)
0.3
8C
16
19
3T
–1
/77
(1.2
9)
–0
.36
––
0.9
6(0
.91
–1
.01
)0
.16
T1
62
09
C2
/64
(3.1
2)
4/7
7(5
.19
)0
.59
(0.1
0–
3.3
5)
0.5
40
.22
(0.0
2–
2.1
6)
0.2
00
.45
(0.0
8–
2.6
4)
0.3
8G
16
21
3A
2/6
4(3
.12
)1
/77
(1.2
9)
2.4
5(0
.21
–2
8.0
5)
0.4
52
.86
(0.2
3–
35
.33
)0
.41
2.6
0(0
.22
–2
9.9
9)
0.4
4C
16
21
4T
–1
/77
(1.2
9)
–0
.36
––
––
A1
62
15
G2
/64
(3.1
2)
––
0.1
1–
––
–A
16
21
9G
1/6
4(1
.56
)6
/77
(7.7
9)
0.1
9(0
.02
–1
.65
)0
.09
0.3
1(0
.03
–2
.97
)0
.31
0.1
9(0
.02
–1
.55
)0
.12
C1
62
22
T–
1/7
7(1
.29
)–
0.3
6–
–0
.19
(0.0
2–
1.5
5)
–T
16
22
4C
2/6
4(3
.12
)2
/77
(2.5
9)
1.2
1(0
.16
–8
.90
)0
.85
1.4
6(0
.08
–2
5.6
2)
0.8
0–
–A
16
23
0G
2/6
4(3
.12
)2
/77
(2.5
9)
1.2
1(0
.16
–8
.90
)0
.85
1.0
4(0
.14
–7
.84
)0
.97
1.5
9(0
.21
–1
2.0
7)
0.6
5T
16
23
1C
–2
/77
(2.5
9)
–0
.19
––
––
C1
62
39
T1
/64
(1.5
6)
1/7
7(1
.29
)1
.21
(0.0
7–
19
.88
)0
.89
––
1.4
8(0
.09
–2
4.8
6)
0.7
9C
16
23
9G
–1
/77
(1.2
9)
–0
.36
––
––
A1
62
40
G–
1/7
7(1
.29
)–
0.3
6–
––
–
(co
nti
nu
ed)
DOI: 10.3109/19401736.2013.836508 Mitochondrial DNA and type 2 diabetes in Tunisia 3
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y M
emor
ial U
nive
rsity
of
New
foun
dlan
d on
07/
14/1
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r pe
rson
al u
se o
nly.
Tab
le2
.C
on
tin
ued
Po
lym
orp
his
ms
T2
Ds
(%)
Co
ntr
ols
(%)
OR
(95
%C
I)p
Val
ue
ORy
(95
%C
I)p
Val
ue
ORz
(95
%C
I)p
Val
ue
A1
62
41
G–
1/7
7(1
.29
)–
0.3
6–
––
–A
16
24
1in
sT1
/64
(1.5
6)
––
0.2
7–
––
–T
16
24
9C
3/6
4(4
.68
)3
/77
(3.8
9)
1.2
1(0
.23
–6
.27
)0
.82
2.4
0(0
.22
–2
6.1
6)
0.4
71
.32
(0.2
6–
6.8
6)
0.7
4C
16
25
6T
3/6
4(4
.68
)1
/77
(1.2
9)
3.7
4(0
.37
–3
7.5
9)
0.2
31
.80
(0.1
4–
22
.80
)0
.65
3.6
2(0
.37
–3
5.7
9)
0.2
7C
16
25
9T
–1
/77
(1.2
9)
–0
.36
––
––
C1
62
60
T1
/64
(1.5
6)
––
0.2
7–
––
–C
16
26
1T
3/6
4(4
.68
)1
/77
(1.2
9)
3.7
4(0
.37
–3
7.5
9)
0.2
3–
–3
.41
(0.3
4–
33
.89
)0
.29
C1
62
64
T3
/64
(4.6
8)
2/7
7(2
.59
)1
.84
(0.2
9–
11
.50
)0
.50
1.2
5(0
.18
–8
.54
)0
.82
1.7
1(0
.28
–1
0.6
5)
0.5
6C
16
27
0T
6/6
4(9
.37
)4
/77
(5.1
9)
1.8
9(0
.50
–7
.07
)0
.34
1.2
3(0
.26
–5
.83
)0
.80
1.9
3(0
.52
–7
.19
)0
.33
T1
62
71
C1
/64
(1.5
6)
2/7
7(2
.59
)0
.59
(0.0
5–
6.7
9)
0.6
70
.63
(0.0
5–
7.6
4)
0.7
20
.51
(0.0
4–
5.8
3)
0.5
9G
16
27
4A
1/6
4(1
.56
)2
/77
(2.5
9)
0.5
9(0
.05
–6
.79
)0
.67
0.7
8(0
.04
–1
3.4
7)
0.8
60
.51
(0.0
4–
5.8
3)
0.5
9C
16
28
7A
1/6
4(1
.56
)1
/76
(1.3
2)
1.1
9(0
.07
–1
9.6
2)
0.9
01
.43
(0.0
8–
24
.56
)0
.80
1.7
1(0
.10
–2
9.4
0)
0.7
1C
16
29
1T
1/6
4(1
.56
)1
/76
(1.3
2)
1.1
9(0
.07
–1
9.6
2)
0.9
00
.52
(0.0
3–
9.2
5)
0.6
61
.01
(0.0
6–
16
.60
)0
.99
C1
62
92
T2
/64
(3.1
3)
5/7
6(6
.58
)0
.46
(0.0
8–
2.4
7)
0.3
50
.26
(0.0
4–
1.6
4)
0.1
50
.49
(0.0
9–
2.6
2)
0.4
0A
16
29
3G
5/6
4(7
.81
)3
/76
(3.9
5)
2.0
6(0
.47
–9
.08
)0
.33
1.1
5(0
.23
–5
.76
)0
.86
2.1
8(0
.50
–9
.60
)0
.30
A1
62
93
T–
1/7
6(1
.32
)–
0.3
6–
––
–C
16
29
5T
–2
/76
(2.6
3)
–0
.19
––
––
C1
62
96
T1
/64
(1.5
6)
3/7
6(3
.95
)0
.39
(0.0
4–
3.8
6)
0.4
00
.23
(0.0
2–
2.6
2)
0.2
40
.31
(0.0
3–
3.0
8)
0.3
2T
16
29
7C
–1
/76
(1.3
2)
–0
.36
––
––
T1
62
98
C5
/64
(7.8
1)
3/7
6(3
.95
)2
.06
(0.4
7–
9.0
8)
0.3
31
.61
(0.3
3–
7.9
9)
0.5
61
.87
(0.4
3–
8.2
4)
0.4
1A
16
30
0G
–1
/76
(1.3
2)
–0
.36
––
––
C1
63
01
T1
/64
(1.5
6)
2/7
6(2
.63
)0
.59
(0.0
5–
6.7
0)
0.6
60
.72
(0.0
6–
8.8
1)
0.8
00
.67
(0.0
6–
7.8
1)
0.7
5T
16
30
4C
4/6
4(6
.25
)5
/76
(6.5
8)
0.9
5(0
.24
–3
.70
)0
.94
0.9
3(0
.21
–4
.07
)0
.93
0.9
4(0
.24
–3
.69
)0
.93
A1
63
09
G5
/64
(7.8
1)
3/7
4(4
.05
)2
.00
(0.4
5–
8.8
3)
0.3
50
.99
(0.2
3–
4.3
3)
0.9
92
.05
(0.4
7–
9.0
0)
0.3
4A
16
31
8T
–3
/74
(4.0
5)
–0
.10
––
––
G1
63
19
A–
3/7
4(4
.05
)–
0.1
0–
––
–C
16
32
0T
2/6
4(3
.13
)4
/74
(5.4
1)
0.5
6(0
.10
–3
.22
)0
.51
0.4
3(0
.07
–2
.61
)0
.36
0.5
7(0
.10
–3
.24
)0
.53
T1
63
24
C–
1/7
4(1
.35
)–
0.3
5–
––
–T
16
32
5C
–1
/74
(1.3
5)
–0
.35
––
––
C1
63
27
T2
/64
(3.1
3)
2/7
4(2
.70
)1
.16
(0.1
6–
8.5
5)
0.8
8–
–1
.20
(0.1
6–
8.8
8)
0.8
6T
16
34
2C
1/6
4(1
.56
)–
–0
.28
––
––
A1
63
43
G2
/64
(3.1
3)
1/7
4(1
.35
)2
.35
(0.2
0–
26
.95
)0
.48
––
2.3
1(0
.20
–2
6.2
2)
0.5
0C
16
35
5T
2/6
4(3
.13
)1
/74
(1.3
5)
2.3
5(0
.20
–2
6.9
5)
0.4
82
.18
(0.1
8–
26
.77
)0
.54
3.4
7(0
.29
–4
1.9
4)
0.3
3T
16
35
6C
1/6
4(1
.56
)–
–0
.28
––
––
C1
63
60
T2
/63
(3.1
7)
1/7
4(1
.35
)2
.39
(0.2
1–
27
.40
)0
.47
0.8
1(0
.05
–1
4.2
8)
0.8
92
.40
(0.2
1–
27
.17
)0
.48
C1
63
61
T–
1/7
4(1
.35
)–
0.3
6–
––
–T
16
36
2C
1/6
3(1
.59
)5
/74
(6.7
6)
0.2
2(0
.02
–2
.01
)0
.14
0.3
6(0
.03
–4
.47
)0
.43
0.2
4(0
.03
–2
.11
)0
.20
T1
63
68
C2
/63
(3.1
7)
––
0.1
2–
––
–G
16
39
0A
10
/63
(15
.87
)4
/74
(5.4
1)
3.3
0(0
.96
–1
1.3
7)
0.0
4*
3.8
3(0
.74
–1
9.9
0)
0.1
13
.33
(0.9
8–
11
.29
)0
.05
C1
63
91
T2
/63
(3.1
7)
1/7
4(1
.35
)2
.39
(0.2
1–
27
.40
)0
.47
––
3.4
6(0
.29
–4
1.2
1)
0.3
3A
16
39
9G
1/6
1(1
.64
)4
/74
(5.4
1)
0.2
9(0
.03
–2
.73
)0
.25
0.1
8(0
.02
–1
.81
)0
.15
0.3
4(0
.04
–3
.23
)0
.35
C1
64
00
T–
1/7
4(1
.35
)–
0.3
6–
––
–
yAd
just
edo
dd
sra
tio
for
age,
sex
and
BM
I;zA
dju
sted
od
ds
rati
ofo
rm
ito
cho
nd
rial
hap
log
rou
ps;
*S
ign
ific
ant
pval
ue
(p5
0.0
5).
4 S. Hsouna et al. Mitochondrial DNA, Early Online: 1–6
Mito
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om b
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emor
ial U
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of
New
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rson
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nly.
An association was found between the G16390A SNP andT2Ds under the univariate model (p¼ 0.04), but the slightlysignificant difference was not detected after multivariate logisticregression analysis with adjustment for age, gender and BMI.The 16390A variant was previously reported in only one study(Isashiki et al., 2003) suggesting that it may be a risk factor forT2Ds onset at older age and other unusual clinical features inJapanese LHON patients with the G11778A variant.
The analysis of the remaining SNPs (87) showed that noneof the association with T2Ds reaches a significant level, even forthe T16189C variant.
This study provides an overview of frequencies distributionof the T16189C variant. It is noteworthy that Tunisian controlsharbor a similar frequency of the 16189C allele (around 30%)as in Asian controls (31%), which is much higher than thatin European individuals (9.2%) (Chinnery et al., 2005).Nevertheless, no association between the T16189C variant andT2Ds was observed in Tunisian population as that reported inAsian studies (Kim et al., 2002; Liao et al., 2008; Park et al.,2008; Weng et al., 2005).
Our findings are in accordance with previous studies thatreported no links between T16189C and T2Ds in Finnish,Scandinavian and British populations (Chinnery et al., 2005;Mohlke et al., 2005; Saxena et al., 2006).
While none of the tested SNPs are associated with T2Ds, oneor more of these variants may influence diabetes and its relatedtraits. Indeed, for a complex disease such as T2Ds, some variantswith modest effect may influence susceptibility in Asian popu-lations (Bhat et al., 2007b; Tang et al., 2006). Most of theseSNPs (82 SNPs) were rare variants. To observe a possible effectof these variants, it would be interesting to study a specific ethnicpopulation rather than a mixed population with large samplesize (Feder et al., 2008). In fact, previous studies performed onEuropean populations with large did not find association betweenmtDNA variants and T2Ds (Chinnery et al., 2005; Mohlke et al.,2005; Saxena et al., 2006).
Our results are in agreement with previous studies investigat-ing the association of susceptibility to T2Ds in Tunisian popula-tions with mitochondrial variant located in tRNALeu gene(Bouhaha et al., 2010; Mkaouar-Rebai et al., 2007).
Conclusions
Except, the weakly association found for the G16390A variant,our results showed that none of the tested variations from theHVS1 region have a major role in T2Ds risk in the studiedTunisian population, and even if such variants were evaluatedwithin a haplogroup context. Since T2Ds is the result ofinteraction between nuclear and mitochondrial genomes aswell as environmental factors, large-scale studies are needed toaccount for these additional factors and shed light on theirinvolvement and role in the physiopathology of T2Ds. It will beinteresting to refine this study, using samples from ethnic specificTunisian and North African populations.
Acknowledgements
We would like to thank the patients and controls for their contributionto this work.
Declaration of interest
The authors declare that no conflicts of interest exist. This work wassupported by the Tunisian Ministry of Public Health, the Ministry ofHigher Education and Scientific Research, the NEPAD/NABNet T2Dproject and EMRO-COMSTECH (3(174)/09-COMSTECH).
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H 23 12 11 0.97T 15 6 9L3 12 5 7L2 11 7 4HV 8 5 3L1 7 5 2U6 7 1 6U5 5 3 2J 5 2 3U 4 1 3K 4 2 2M1 4 2 2L0 3 1 2W 3 1 2I 2 1 1M 1 1 0X 1 1 0R0 1 1 0Missingy 25 7 18
yMissing haplogroups; zThe difference in haplogroup distributionbetween T2Ds and control groups was statistically tested throughFisher’s exact probability test on 10� 2 contingency table (haplogroupcategories less than five individuals were pooled into one group).
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