DNA G7444A, 2. Study on a new point mutation of nt7444 G A in the mitochondrial DNA in a type 2 diabetes mellitus family

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1 HEREDITAS (Beijing) , 29(4): ISSN DOI: /yc DNA G7444A,,,,,,,, : PCR-RFLP 2 DNA G7444A,, 27, 11 DNA G7444A, , 1,, DNA G7444A, 2 : DNA; 2 ; G7444A Study on a new point mutation of nt7444 G A in the mitochondrial DNA in a type 2 diabetes mellitus family CHENG Zu-Jian, YANG Bin, LIU Qi-Cai, JIANG Ling, XIE Hai-Hua, OU Qi-Shui Depertment of Laboratory Medicine, The First Affiliated Hospital, Fujian Medical University, Fuzhou , China Abstract: Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and direct sequencing were applied to detect a new point mutation of nt7444g A in the mitochondrial DNA in a type 2 diabetes mellitus family. The related clinical data were also collected and analyzed. mtdna G7444A mutation in the cytochrome c oxidase I (COI) gene was found in 11 of 27 cases, all of whom were from the maternal side. Among them, 5 were confirmed to have type 2 diabetes mellitus, and one had impaired glucose tolerance. We conclude that the novel point mutation of mtdna G7444A may be an independent factor associated with type 2 diabetes mellitus. Keywords: mitochondrial DNA; type 2 diabetes mellitus; nt7444 G A mutation (diabetes mellitus, DM), DNA(mitochondrial DNA, mtdna),, [1] DNA, nt3243a G, trna Leu (UUR) 10, nt3252t C nt3250c T nt3256c T nt3260a G 3316 G A [2,3] 2 DNA, DNA COI 7444 G A : ; : : ( : FJGXY04005) [Supported by Science Development Foundation of Fujian Medical University (No. FJGXY04005)] : (1974 ),,,, : Tel: ; chengzujian@163.com : (1970 ),,,,, : Tel: ; ouqishui@163.com

2 434 HEREDITAS (Beijing) , 4 27 ( 1) 5, ; 1 ( 3); ( 5),, 55, 2 15, 2, 3,, 2 27, 17 ( 11, 6 ), , 8 h, 126 mg/dl (7.0 mmol/l) 200 mg/dl (11.1 mmol/l) DNA, 5 ml Wizard Genomic DNA Purification Kit (Promega ) DNA, PCR PCR Primer Express, : 5 -gctacaccctagaccaaaccta-3 ; : 5 - ccgtagtcggtgtactcgta-3 ( ) PCR 25 µl, 50 ng DNA, 0.06 mol/l, 0.2 mmol/l dntp, 10 PCR 2.5 µl, 2.0 mmol/l Mg 2+, 1 U Taq (Promega ) PE min, s s s, 35, min 1.5% PCR (1) PCR : Promega Wizard DNA clean-up system kit, ; (2) : PCR 10 µl Xba (Promega )5 U, 37 2 h 1.5%, PCR PCR DNA C, ( ) : (GLU)( ), (LAC)( ), (TG LDL)( ), C( ) PCR-RFLP Xba 5 TCTAGA 3,, Xba PCR, Xba 818 bp 1, 482 bp 336 bp bp 336 bp, ; bp, ( 2) 2.2 DNA PCR DNA NCBI mtdna (NC ), Xba mtdna G7444A ( 3) 1 2 : ; : 2 ; : ; : mtdna G7444A Fig. 1 Pedigree of the family with type 2 diabetes mellitus : Indicates the proband ; : Type 2 Diabetes Mellitus; : Normal; : Obligate carrier.

3 4 : 2 DNA G7444A mtdna 7444 PCR (Xba ) M: Marker; 1 4, 6 7: mtdna 7444 PCR ; 5: mtdna 7444 PCR Fig. 2 Agarose gel electrophoresis results of PCR products containing mtdna 7444 site cut by Xba M: 100 bp ladder; 1 4, 6, 7: PCR products with wildtype mtdna7444 cut by Xba ; 5: PCR product with mutated mtdna7444 cut by Xba 2.3 1, mt DNA G7444A,, ATP DNA,, mtdna (MIDD) (DM), :(1) : mtdna G7444A, ; (2) : , 1 ; (3) DM : 5 mtdna G7444A DM 45.5, ; (4) : mtdna G7444A 3 mtdna 7444 PCR A: G mtdna 7444 ; B: A mtdna 7444 (G A) Fig. 3 Sequence of PCR products containing mtdna 7444 A: Normal control, the arrow indicates the wildtype nucleotide; B: A patient of the pedigree family, with the arrow indicating the mutated nucleotide (G A).

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5 4 : 2 DNA G7444A 437 Brown [4] mtdna G7444A Leber (LHON), mtdna G7444A 2 mtdna G7444A, 5 mtdna G7444A,, 40 mtdna G7444A, COI mtdna 7444 VI( C, COI), COI AGA AAA, COI COI C 3 ( - - ),, C,, [4] DNA,, ATP, β ATP K +,, [5] Guan [6] trna ser(ucn ) A7445G trna ser(ucn), trna ser(ucn ) ND6 rrna, 7445 mtdna G7444A (References): [1] Maassen JA, Van Essen E, Van den Ouweland JM, Lemkes HH. Molecular and clinical aspects of mitochondrial diabetes mellitus. Exp Clin Endocrinol Diabetes, 2001, 109(3) [2] Tawata M, Ohtaka M, Iwase E, Ikegishi Y, Aida K, Onava T. New mitochondrial DNA homoplasmic mutation associated with Japanese patient with type 2 diabetes. Diabetes, 1998, 47(2): [3] ZHOU Xiao-Lei, ZHANG Li-Shan, HUANG Ying, TIAN Cheng-Gong, QIU Ding-Hong, LU Ming-Hua, ZHANG Zhi-Ping. Mitochondrial DNA mutation associated with NIDDM. Hereditas (Beijing), 1997, 19(2): 5 8.,,,,,,. NIDDM., 1997, 19(2): 5 8. [4] Brown MD, Yang CC, Trounce I, Torroni A, Lott MT, Wallace DC. A mitochondrial DNA variant, identified in Leber hereditary optic neuropathy patients, which extends the amino acid sequence of cytochrome coxidase subunit I. Am J Hum Genet, 1992, 51(2): [5] Maassen JA, Thart LM, Van Essen E, Heine RJ, Nijpels G, Jahangir Tafrechi RS, Raap AK, Janssen GM, Lemkes HH. Mitochondrial diabetes: molecular mechanisms and clinical presentation. Diabetes, 2004, 53: [6] Guan MX, Enriquez JA, Fischel-Ghodsian N, Puranam RS, Lin CP, Maw MA, Attardi G. The deafness associated mtdna 7445 mutation, which affects trna Ser(UCN) precursor processing, has long-range effects on NADH dehydrogenase ND6 subunit gene expression. Mol Cell Biol, 1998, 18(10): ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ, : α -1, : - -SEA/αα (1) α,?? (2) 20,,,? (3),? (1) α ( ) α -1 α (2) (MCV) (Hb) α 10% α (3) α 1/4 α α 16~20 ( : )