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Introduction

Metabolic syndrome (1) is a complex disorder, characterized by a group of metabolic disorders, including hyperglycemia, hypertension, hyperlipaemia and central obesity, which are the risk factors of cardiovascular disease and diabetes. There is a high prevalence of MS in developed countries. According to the national health statistic reports issued in 2009 from the National Health and Nutrition Examination Survey 2003–2006, the prevalence of metabolic syndrome in the USA, is 34% in the population of 20 years of age. The prevalence of metabolic syndrome increased in different age groups for the two genders (2). However, in recent years, the prevalence of MS has increased at an alarming rate in developing countries and districts of Asia (3–5). According to the National Health and Nutrition Examination Survey 2003–2006, Chinese prevalence of MS (4) increased by 14–18% in 2005 and has continued to rise. Approximately 60–80% of diabetic patients are also MS patients.

An unhealthy lifestyle, including lack of physical exercise, bad nutritional habits, hormone changes and aging factors are associated with the onset of MS (6). However the two important risk factors for MS are central obesity and insulin resistance (7). Individuals with excess abdominal fat are more likely to develop hypertension and increased levels of blood cholesterol, triglycerides and fatty acids, which may affect insulin in glucose regulation (7) and lead to insulin resistance.

MS is caused by environmental and genetic factors. Previous studies have revealed numerous target molecules and variation associated with MS. Nuclear genes, including peroxisome proliferator-activated receptor-γ, lamin A/C gene and IL-6, are considered as susceptibility genes of MS (8–12). Genetic factors, including mitochondrial DNA variants have also been associated with the onset of MS (1,13,14).

Mitochondrial DNA (mtDNA) (15) contains 37 genes, including 2 ribosomal RNA genes, 22 transfer RNA genes and 13 coding genes, which code the proteins involved in oxidative phosphorylation. mtDNA has a higher mutation rate than nuclear DNA as it lacks protective histones and is susceptible to oxidative damage from reactive oxygen species (ROS) generated during respiration in the mitochondria (16). Studies have indicated that mitochondrial genetic defects may lead to β-cell dysfunction and insulin resistance (17). Moreover, mitochondrial dysfunction and biogenesis may be involved in the development of MS (18,19).

In the present study, a juvenile-onset metabolic syndrome male with a maternally inherited diabetes family was examined. In the patient’s family, a number of members diagnosed with type 2 diabetes also presented characteristics of MS, however, the 18-year-old male had not developed diabetes. Notably, a heteroplasmic mtDNA mutation, A8890G, observed in the male was not observed in other family members. The A8890G is located in the ATPase 6 gene, which encodes for a subunit of ATP synthase (ATPase) in mitochondria. Therefore, it is hypothesized that A8890G may be associated with mitochondrial dysfunction and may contribute to juvenile-onset of MS.

Materials and methods

Subjects

A maternally inherited diabetes family was contacted through the Endocrinology Department and the Medical Examination Center of the First Affiliated Hospital of Wenzhou Medical College of Zhejiang Province in China and recruited. Peripheral blood samples were obtained from the patient and all participating family members. Serum triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), glucose, liver profile and kidney function parameters were determined using routine automated assay methods following an overnight fast.

Diagnosis of MS was determined according to the diagnostic criteria proposed by the International Diabetes Federation (IDF) in 2005 (6). Subjects were diagnosed with metabolic syndrome if they had a waist circumference of >90 cm in males and >80 cm in females and two or more of the following four components: i) serum triglycerides of >1.7 mmol/l or having clinical treatment; ii) HDL-cholesterol levels <0.9 mmol/l in males and <1.0 mmol/l in females or having clinical treatment; iii) BP of ≥130/85 mmHg or under anti-hypertensive treatment; iv) fasting glucose of ≥5.6 mmol/l or known treatment for diabetes. Subjects with ≤2 risk components were excluded.

A total of 165 freshmen were recruited in Wenzhou Medical College and underwent a routine healthy check-up. The study was approved by the Hospital Ethics Committee. Informed consent was obtained from all participating subjects.

Mitochondrial DNA analysis

Genomic DNA was extracted from peripheral blood leukocytes of each individual using universal genomic DNA extraction kit version 3.0 (Takara Bio Inc., Shiga, Japan). Entire mitochondrial DNA was amplified in 24 overlapping fragments using primer sets, as described in a previous study (20).

PCR was performed at a final volume of 22 μl as follows: 1.5 μl MgCl2 (25 mmol/l), 2.0 μl dNTP mixture (2.5 mmol/l of each component), 2.0 μl 10X Ex Taq buffer, 0.2 μl of each primer (10 μmol/l), 0.1 μl Ex Taq polymerase (Takara Bio Inc.) and 2.0 μl genomic DNA as the template. PCR was performed in a MyCycler Thermal Cycler 170–9703 (Bio-Rad, Hercules, CA, USA).

PCR conditions were as follows: initial denaturation at 94ºC for 5 min, followed by 35 cycles of denaturation at 94ºC for 30 sec, annealing at 60ºC for 45 sec and extension at 72ºC for 1 min, with a final extension at 72ºC for 6 min. Amplification products were confirmed using electrophoresis in 1.2% agarose gels and visualized by staining with ethidium bromide. PCR products underwent direct sequencing in an ABI 3730 DNA sequencer (Sigma, St. Louis, MO, USA). Sample sequences were compared with the revised Cambridge Reference Sequence (rCRS) from Mitomap, a human mitochondrial genome database (http://www.mitomap.org).

Multiple amino acid sequence alignment was performed by Clustal X. The secondary structure of mitochondrial ATPase6 was predicted by the SOSUI system (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html).

Denaturing high-performance liquid chromatography (DHPLC) assay

PCR amplification of mtDNA mt 8702–8982 (281 bp) was performed at a final volume of 20 μl as follows: 0.1 μl Pyrobest DNA Polymerase (Takara Bio Inc.), 2.0 μl 10X Pyrobest buffer (containing Mg2+), 3.0 μl dNTP mixture, 0.5 μl of each primer (10 μmol/l, DHPLC grade), 2.0 μl genomic DNA as template. The primer sequences used were: forward: 5′-CCATACACAACACTAAAGGACGAA-3′ and reverse: 5′-TTGAATGAGTAGGCTGATGGTTT-3′. PCR conditions were as follows: an initial denaturation at 95ºC for 5 min, followed by 35 cycles of denaturation at 95ºC for 10 sec, annealing at 60ºC for 20 sec and extension at 72ºC for 45 sec, with a final extension at 72ºC for 6 min. PCR products were denatured at 95ºC for 1 min and cooled to 4ºC at a rate of 1ºC/min to allow for heteroduplex formation. The DHPLC assay was performed in a WAVE® nucleic acid fragment system (Transgenomic Inc., Omaha, NE, USA).

Results

Family and biochemical analysis

Clinical and biochemical characteristics of all the family members are provided in Table I. The patient details presented are of a typical maternally inherited diabetes family (Fig 1A).

Figure 1 Pedigree chart and mtDNA analysis of the family. (A) Pedigree chart of the family. Arrow denotes proband. (B) A8890G mutation in III3 mitochondrial ATP6 gene. The left and right are sequencing and anti-sequencing results separately, the arrow denotes the variation site. (C) Agarose gel electrophoresis of PCR products (mt 8702–8982, 281 bp), M: DNA marker DL2000; lanes 1–6: PCR products 281 bp. (D) DHPLC analysis result of III3. Spectrum of III3 shows two peaks, the arrow denotes a heterologous double chain peak, the percentage of the peak area is 33.261 and 66.739%, respectively; 10-III3 is the patient, 294 is the negative control. (E) DHPLC analysis results of other family members, 294 is the negative control.

Table I Clinical and biochemical characteristics of all family members.

Table I

Clinical and biochemical characteristics of all family members.

Variables	Family members
General	I2	II1	II4	II5	II6	III1	III2	III3
Gender	F	M	F	M	F	F	M	M
Age, years	85	61	54	45	43	38	33	18
Waist circumstances, cm	75	97	87	85	90	94	87	95
BMI (kg/m2)	20.02	28.08	24.30	21.45	26.35	27.34	21.71	27.47
DBP (mmHg)	62	80	78	75	80	80	70	70
SBP (mmHg)	120	130	126	130	120	130	110	110
History of diabetes, years	10	10	4	1	1	0	0	0
Biochemical parameters
FBG (mmol/l)	4.8	7.78	5.17	6.98	10.78	5.87	5.08	5.08
HbA1c1 (%)	6.58	7.57	6.01	7.81	9.39	5.1	5.35	4.65
BUN (mmol/l)	5.7	4.1	6.9	4.3	3.9	6.1	4.3	4
Cr (μmol/l)	65	75	81	60	40	50	54	51
UA (μmol/l)	447	346	409	307	214	288	230	342
Cys C (mg/l)	1.41	1.32	1.57	1.28	0.68	1.20	1.09	1.29
HB (mmol/l)	0.07	0.06	0.07	0.05	0.12	0.09	0.06	0.07
Lipid parameters
TC (mmol/l)	4.15	4.24	4.58	7.33	5.01	6.29	4.54	6.78
TG (mmol/l)	0.54	1.18	1.1	2.43	2.22	1.56	1.55	1.8
HDL-C (mmol/l)	1.4	1.3	1.36	1.24	0.96	1.59	1.04	0.97
LDL-C (mmol/l)	2.27	2.64	2.62	4.79	2.52	3.96	2.74	4.86
apoA-I (g/l)	1.2	1.38	1.41	1.07	1.24	1.53	0.94	1.13
apoB-100 (g/l)	0.98	1.15	1.13	1.62	1.11	1.29	0.93	2.14
apoA-I/apoB-100	1.22	1.2	1.25	0.66	1.12	1.19	1.01	0.53
Hearing loss	+	+	+
			(Single ear)	−	−	−	−	−

[i] DBP, diastolic arterial blood pressure; SBP, systolic arterial blood pressure; FBG, fasting blood glucose; BUN, blood urine nitrogen; Cr, creatine; UA, urine acid; Cys C, cystine C; HB, β-hydroxybutyric acid; TC, total cholesterol; TG, triglycerol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

According to the IDF MS diagnostic criteria (21,22), the 18-year-old male (III3) was diagnosed with MS and other family members II1, II5, III6, III1 and III3 also presented MS features. Subjects I2, II1, II4, II5 and II6 suffered from diabetes for >1 year, with hearing loss and mild kidney impairment. Subjects II1, II6, III1 and III3 were overweight (BMI 25–29 kg/cm2, Table I). No obesity (BMI≥30) and hypertension was observed in the family. In addition, it was observed that the male patient (III3) and the patient’s father (II5) presented lipid metabolism dysfunction with higher TG and LDL-C levels, lower HDL-C level and <1.0 of the ApoA-I/ApoB100 ratio (Table I).

mtDNA variant analysis

Mitochondrial genomic DNA variants in the family are provided in Table II. An MS-associated variant T16189C was observed in this family. Using sense-, antisense-sequencing and DHPLC analysis, a heteroplasmic A-G substitution at mt 8890 (Fig. 1B and C) was detected only in the 18-year-old male patient (III3). A8890G changed the basic amino acid Lys to Glu at position 122 of the ATPase6 subunit. This amino acid is located in the intermembrane of the mitochondria (Fig. 2) and is highly conserved in 30 species (Table III). In addition, there are numerous mtDNA variants, however A8890G was not observed on the mitochondrial ATPase 6 gene of the control subjects. No other MS or diabetes-associated mutations were detected in this patient or the patient’s family.

Figure 2 Secondary structure prediction of ATPase6. Arrow denotes Lys122 in inter-membrane region.(http://bp.nuap.nagoya-u.ac.jp/sosui/cgi-bin/adv_sosui.cgi).

Table II mtDNA variants in this family.a

Table II

mtDNA variants in this family.a

Gene	Position	Nucleotide replacement	Amino acid replacement	Conservation (H/B/M/X)b	Previously Reportedc
D-Loop	73	A-G			Y
	263	A-G			Y
	315	C-CC			Y
	489	T-C			Y
	16184	C-T			Y
	16189	T-C			Y
	16223	C-T			Y
	16298	T-C			Y
	16319	G-A			Y
12S rRNA	750	A-G		A/A/A/-	Y
	1438	A-G		A/A/A/G	Y
16S rRNA	2706	A-G		A/G/A/A	Y
	2835	C-T		C/A/A/A	Y
ND2	4715	A-G			Y
	4769	A-G			Y
CO1	6179	G-A			Y
	7028	C-T			Y
	7196	C-A			Y
CO2	8245	A-G			Y
ATP6	8860	A-G	Thr-Ala	T/A/A/T	Y
	8890	A-G	Lys-Glu	K/K/K/N	N
	9053	G-A	Ser-Asn	S/G/G/T	Y
CO3	9540	T-C			Y
ND3	10398	A-G	Thr-Ala	T/T/T/A	Y
	10400	C-T			Y
ND4	10873	T-C			Y
	11176	G-A			Y
	11719	G-A			Y
ND5	12705	C-T			Y
ND6	14470	T-C			Y
CytB	15043	G-A			Y
	15301	G-A			Y
	15326	A-G	Thr-Ala	T/M/I/I	Y
	15487	A-T		P/P/P/P	Y

a Family members include III3 and all maternal relatives;

b H: Homo sapiens; B: Bos taurus; M: Mus musculus; X: Xenopus laevis;

c Y, yes; N, no.

Table III Amino acid in ATPase6 position 22 of 30 species.

Table III

Amino acid in ATPase6 position 22 of 30 species.

Species	Aminoacid
Gorilla gorilla	Lys
Pan paniscus	Lys
Pan troglodytes	Lys
Pongo pygmaeus	Lys
Hylobates lar	Lys
Papio hamadryas	Lys
Equus caballus	Lys
Equus asinus	Lys
Rhinoceros unicornis	Lys
Ceratotherium simum	Lys
Bos taurus	Lys
Ovis aries	Lys
Sus scrofa	Lys
Balaenoptera musculus	Lys
Balaenoptera physalus	Lys
Hippopotamus amphibius	Lys
Mus musculus	Lys
Rattus norvegicus	Lys
Myoxus glis	Asn
Oryctolagus cuniculus	Lys
Felis catus	Lys
Halichoerus grypus	Lys
Phoca vitulina	Lys
Canis familiaris	Lys
Artibeus jamaicensis	Lys
Dasypus novemcinctus	Lys
Didelphis virginiana	Lys
Macropus robustus	Lys
Ornithorhyncus anatinus	Lys
Xenopus laevis	Asn

Discussion

Although nuclear genes are involved in MS onset, mtDNA mutations are also significant in the development of MS. Mitochondrial tRNAIle T4291C mutation was first observed in a Caucasian population with MS where it was hypothesized to cause a cluster of metabolic defects. In addition, mtDNA D-loop T16189C was widely implicated in the development of insulin resistance, MS and coronary artery disease (14,23). Subsequent studies confirmed that mutations in mtDNA were associated with diabetes, particularly, heteroplasmy tRNALeu(UUR) A3243G was implicated in causing maternal-inherited diabetes (24–26). Extensive studies on the A3243G mutation revealed inefficient aminoacylation, impairments in the processing of tRNA precursors and base post-transcriptional modification of tRNALeu(UUR), which induced mitochondrial dysfunction (27–31).

In the present study, a juvenile-onset metabolic syndrome male in a maternally inherited diabetes family was examined. Although the family was a typical diabetic family, characteristics of MS with mild increasing cystine C levels, a sensitive marker for renal impairment, were also presented. The patient presented central obesity (overweight and 95 cm of waist circumference), dyslipidemia (high TG, low HDL level and low apoA-I/apoB-100) and mild renal impairment according to cystine C levels. Obesity is a risk factor for insulin resistance and pancreatic β-cells are likely to secrete more insulin when compensating for insulin resistance. The dysfunction of β-cells may cause pre-diabetes, a condition ultimately leading to diabetes (32).

Notably, the mtDNA genome assay detected a heteroplasmic mtDNA mutation A8890G in the patient and was not observed in other members of the family. In addition, MS-associated mitochondrial T16189C single-nucleotide polymorphism was detected in all the family members. No known diabetes or MS-associated mitochondrial mutations, including tRNALeu(UUR)A3243G and tRNAIleT4291C (1), were detected.

A8890G is located in ATPase 6 gene, which encodes for a subunit of ATP synthase in mitochondria. ATPase is also known as mitochondrial respiratory chain complex V. It is a key enzyme in cell energy conversion, participating in the synthesis and hydrolysis of ATP. ATPase6 is one of the ATPase subunits and is coded by the mitochondrial ATP6 gene. ATPase6 is a component of the proton channel and is essential for proton transportation and energy production (33,34). According to the mitomap database, http://mitomap.org, A8890G has not been previously reported. The majority of frequently reported mutations of ATPase 6 gene were heteroplasmic transversion T8993C and T8993G, which were associated with Leigh and NARP syndrome. Symptom severity was proportional to the heteroplasmy load (35–38). Cell and mitochondrial function analysis indicated that mutations led to energy deprivation, ROS overproduction and reduced ATP production (39–41).

Unlike T8993C/G (Leu156Pro/Arg), which changed an amino acid in the mitochondrial transmembrane protein, A8890G changed the amino acid in the mitochondrial intermembrane protein, which may affect the electrochemical proton gradient and energy production. These changes may contribute to the development of MS. Further evaluation of this abnormality is likely to include tissue biopsy, mitochondrial function tests and cell analysis.

This mutation was not detected in other family members, possibly as this mutation is a somatic mutation or a new germline mutation. Mitochondrial replicative segregation during cell division may affect the mutation (42). This indicates that the patient’s mother (II6) may be a mosaic for this mutation. A low level of A8890G mutant mitochondria that cannot be detected using present analytical methods may be present in the patient’s mother. At pre-ovulatory oocyte division, mutant and normal mitochondria segregate into next generation oocytes at random, producing uneven loading of mutant mitochondria. The patient may originate from an oocyte with a higher heteroplasmy rate.

In conclusion, the novel heteroplasmic A8890G may contribute to juvenile-onset MS. This mutation has not been detected in other family members, possibly as it is a somatic mutation or due to production of a new germline mutation or mitochondrial genetic segregation in the juvenile-onset patient.

Acknowledgements

Thanks for all the family members. This study is supported by the National Key Technology R&D Program of China (Grant no. 2009BAI80B02); the Science Foundation of Zhejiang Province grants (nos. Y2090753, Y2100582); Zhejiang Provincial Education Department Research grant (no. Y200804470).

Abbreviations:

References