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. 2004 May;2(2):99-106.
doi: 10.3121/cmr.2.2.99.

Genotyping Parkinson disease-associated mitochondrial polymorphisms

Yiguo Jiang  1 , Tammy Ellis, Anne R Greenlee
Affiliations

Genotyping Parkinson disease-associated mitochondrial polymorphisms

Yiguo Jiang et al. Clin Med Res. 2004 May.

Abstract

Objective: The purpose of this study was to establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (T(m)) analysis. This technology should prove useful for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson disease (PD).

Methods: Mitochondrial DNA (mtDNA) was extracted from whole blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler (Roche Molecular Biochemical, Mannheim, Germany). Genotyping of 10 SNPs in 15 subjects was based on the analysis of allele-specific T(m) of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products.

Results: Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The nucleotide changes at positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, and 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, and 9.52 degrees C declines in T(m) of the detection probes, respectively. Genotyping of all 150 samples was verified by 100% correspondence with the results of sequencing. Fourteen subjects were haplogrouped by combining results for all 10 SNPs.

Conclusion: A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes was developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies, and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.

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Figures

Figure 1
Figure 1
Orientation of mitochondrial DNA, PCR primers, amplicon, and the fluorophore-labeled hybridization probes for SNP C7028T. This probe corresponds to the mutation. The polymorphism at nucleotide 7028 is the result of a C to T substitution, which creates an A–T match between the antisense strand and the sensor probe. Complete matching of sensor probe to the antisense strand results in a higher Tm of the hybrid. The mismatch destabilizes the hybrid such that a decrease in the probe Tm occurs.
Figure 2
Figure 2
PCR amplification and genotyping of C7028T SNP using hybridization probes and melting curve analysis. (A) Plot of fluorescence intensity signal (F2) versus cycle number for SNP 7028. The 194 bp fragment which covers the 7028 site was amplified from genomic mtDNA of two different genotypes, either 7028C allele (curve I) or 7028T allele (curve II). The no-template control (curve III) shows no amplification. (B) Following amplification a melting analysis of amplified fragments was immediately performed. Data for the plot was obtained during the melting transition of the probe from the amplified fragment. The melting curves were plotted for a sample for the 7028C allele (curve I) and a sample for the 7028T allele (curve II). Melting analysis of a no-template control (curve III) was also performed. The melting peaks indicate that the 7028C allele sequence has a lower Tm than the 7028T allele sequence due to homology between the probe and mutant allele.
Figure 2
Figure 2
PCR amplification and genotyping of C7028T SNP using hybridization probes and melting curve analysis. (A) Plot of fluorescence intensity signal (F2) versus cycle number for SNP 7028. The 194 bp fragment which covers the 7028 site was amplified from genomic mtDNA of two different genotypes, either 7028C allele (curve I) or 7028T allele (curve II). The no-template control (curve III) shows no amplification. (B) Following amplification a melting analysis of amplified fragments was immediately performed. Data for the plot was obtained during the melting transition of the probe from the amplified fragment. The melting curves were plotted for a sample for the 7028C allele (curve I) and a sample for the 7028T allele (curve II). Melting analysis of a no-template control (curve III) was also performed. The melting peaks indicate that the 7028C allele sequence has a lower Tm than the 7028T allele sequence due to homology between the probe and mutant allele.
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