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2014 | 9 | 6 | 839-848
Tytuł artykułu

Sensitive quantification of mitochondrial mutation using new Taqman probes

Treść / Zawartość
Warianty tytułu
Języki publikacji
EN
Abstrakty
EN
The A3243G mitochondrial mutation is the major cause of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The severity of the disease is correlated with the heteroplasmy level of the mutation. Here we describe for the first time the validation of a real-time polymerase chain reaction (PCR) assay with Taqman locked nucleic acid (LNA) fluorescent (FAM for mutant, HEX for wild type) probes for quantification of heteroplasmy levels in a total of 18 family members from 5 Vietnamese MELAS patients carrying A3243G. Almost no background of FAM signals was detected in normal samples, indicating that the probes were allele-specific. Standard curves indicate sensitive detection at 0.1% mutants and high reliability with R2 > 0.985. The correlation line between measured % mutant and expected % mutant was highly reliable, with a slope of 0.993 and R2 of 0.998. All positive A3243G mutant samples pre-screened by PCR-restriction fragment length polymorphism (RFLP) were confirmed, and their heteroplasmy levels quantified to be from 3.68 to 80.85%. The heteroplasmy levels in patients were higher than in their family members and generally correlated well with the severity of their clinical symptoms. Overall, this work is the first demonstration of the application of LNA probes for sensitive and highly reliable quantification of heteroplasmy levels in human mitochondria.
Wydawca

Czasopismo
Rocznik
Tom
9
Numer
6
Strony
839-848
Opis fizyczny
Daty
wydano
2014-12-01
online
2014-08-16
Twórcy
autor
  • Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai Street, Thanhxuan, Hanoi, Vietnam
  • Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai Street, Thanhxuan, Hanoi, Vietnam
  • Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai Street, Thanhxuan, Hanoi, Vietnam
autor
  • Department of Neurology, National Hospital for Pediatrics, 18/879 De Lathanh Street, Dongda, Hanoi, Vietnam
  • Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen Trai Street, Thanhxuan, Hanoi, Vietnam, phantuannghia@vnu.edu.vn
Bibliografia
  • [1] Masakazu M., Hideyuki H., Takashi I., Hiroshi I., Susumu F., Manami A., et al., Different effects of novel mtDNA G3242A and G3244A base changes adjacent to a common A3243G mutation in patients with mitochondrial disorders. Mitochondrion, 2009, 9, 115–122 http://dx.doi.org/10.1016/j.mito.2009.01.005[Crossref][WoS]
  • [2] Bai R.K., Wong L.J.C., Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach. Clin. Chem., 2004, 50, 996–1001 http://dx.doi.org/10.1373/clinchem.2004.031153[Crossref]
  • [3] Ma Y., Fang F., Yang Y., Zou L., Zhang Y., Wang S., et al., The study of mitochondrial A3243G mutation in different samples. Mitochondrion, 2009, 9, 139–143 http://dx.doi.org/10.1016/j.mito.2009.01.004[Crossref][WoS]
  • [4] Smith M.L., Hua X.Y., Marsden D.L., Liu D., Kennaway N.G., et al., Diabetes and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS): radiolabeled polymerase chain reaction is necessary for accurate detection of low percentages of mutation. J. Clin. Endocrinol. Metab., 1997, 82, 2826–2831
  • [5] Tsukuda K., Suzuki Y., Kameoka K., Osawa N., Goto Y.I., Katagiri H., et al., Screening of patients with maternally transmitted diabetes for mitochondrial gene mutations in the tRNA Leu(UUR) region. Diabet. Med., 1997, 14, 1032–1037 http://dx.doi.org/10.1002/(SICI)1096-9136(199712)14:12<1032::AID-DIA504>3.0.CO;2-Y[Crossref]
  • [6] White H.E., Durston V.J., Seller A., Fratter C., Harvey J.F., Cross N., Accurate detection and quantitation of heteroplasmic mitochondrial point mutation by pyrosequencing. Genet. Test., 2005, 9, 190–199 http://dx.doi.org/10.1089/gte.2005.9.190[Crossref]
  • [7] Urata M., Wakiyama M., Iwase M., Yoneda M., Kinoshita S., Hamasaki N., et al., New sensitive method for the detection of the A3243G mutation of human mitochondrial deoxyribonucleic acid in diabetes mellitus patients by ligation-mediated polymerase chain reaction. Clin. Chem., 1998, 44, 2088–2093
  • [8] Tajima H., Sueoka K., Moon S.Y., Nakabayashi A., Sakurai T., Murakoshi Y., et al., The development of novel quantification assay for mitochondrial DNA heteroplasmy aimed at preimplantation genetic diagnosis. J. Assist. Reprod. Genet., 2007, 24, 227–232 http://dx.doi.org/10.1007/s10815-007-9114-0[Crossref][WoS]
  • [9] Singh R., Ellard S., Hattersley A., Harries L.W., Rapid and sensitive real-time polymerase chain reaction method for detection and quantification of 3243A-G mitochondrial point mutation. J. Mol. Diagn., 2006, 8, 225–230 http://dx.doi.org/10.2353/jmoldx.2006.050067[Crossref]
  • [10] Souza A.C., Ferreira R.C., Gonçalves S.S., Quindós G., Eraso E., Bizerra F.C., et al., Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR. J. Clin. Microbiol. 2012, 50, 2310–2314 http://dx.doi.org/10.1128/JCM.00303-12[WoS][Crossref]
  • [11] Li Q., Yuan Y.Y., Huang D.L., Han D.Y., Dai P., Rapid screening for the mitochondrial DNA C1494T mutation in a deaf population in China using realtime quantitative PCR. Acta Otolaryngol., 2012, 132, 814–818
  • [12] Wang J.Y., Gu Y.S., Wang J., Tong Y., Wang Y., Shao J.B., et al., MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR. J. Zhejiang Univ. Sci. B, 2008, 9, 610–615 http://dx.doi.org/10.1631/jzus.B0820058[Crossref]
  • [13] Trinh L.P., Chu V.M., Phan T.N., Detection of point mutation A3243G of MELAS syndrome using modified PCR-RFLP. J. Genet. Appl. 2009, 4, 6–9, (in Vietnamese)
  • [14] Letertre C., Perelle S., Dilasser F., Arar K., Fach P., Evaluation of the performance of LNA and MGB probes in 5’ nuclease PCR assays. Mol. Cell Probes 2003, 17, 307–311 http://dx.doi.org/10.1016/j.mcp.2003.08.004
Typ dokumentu
Bibliografia
Identyfikatory
Identyfikator YADDA
bwmeta1.element.-psjd-doi-10_2478_s11536-013-0325-8
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