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. 2010 Aug;14(4):571-6.
doi: 10.1089/gtmb.2010.0030.

A Single-Tube Quantitative High-Resolution Melting Curve Method for Parent-Of-Origin Determination of 15q Duplications

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A Single-Tube Quantitative High-Resolution Melting Curve Method for Parent-Of-Origin Determination of 15q Duplications

Nora Urraca et al. Genet Test Mol Biomarkers. .
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The most common chromosomal abnormalities associated with autism are 15q11-q13 duplications. Maternally derived or inherited duplications of 15q pose a substantial risk for an autism phenotype, while paternally derived duplications may be incompletely penetrant or result in other neurodevelopmental problems. Therefore, the determination of maternal versus paternal origin of this duplication is important for early intervention therapies and for appropriate genetic counseling to the families. We adapted a previous single-reaction tube assay (high-resolution melting curve analysis) to determine the parent of origin of 15q duplications in 28 interstitial duplication 15q samples, one family and two isodicentric subjects. Our method distinguished parent origin in 92% of the independent samples as well as in the familial inherited duplication and in the two isodicentric samples. This method accurately determines parental origin of the duplicated segment and measures the dosage of these alleles in the sample. In addition, it can be performed on samples where parental DNA is not available for microsatellite analysis. The development of this single-tube assay will make it easier for genetic testing laboratories to provide parent-of-origin information and will provide important information to clinical geneticists about autism risk in these individuals.


FIG. 1.
FIG. 1.
SNRPN quantitative HRM analysis in control samples. The colors indicate HRM analysis in AS deletion (red), PWS UPD (dark green), and a nonduplication control sample (gray). (a) Melting peaks indicate one peak at ∼81.7°C for the AS deletion sample that has no maternal allele and one peak at ∼86.2°C for PWS UPD that has no paternal allele. The normal control shows two peaks of approximate equal intensity at ∼81.7°C for the paternal allele and ∼86.2°C for the maternal allele. (b) The normalized melting profiles indicate that 100% of the relative signal intensity comes from the ∼81.7°C melting curve in the AS deletion sample, while 100% of the signal comes from the ∼86.2°C melting curve in the PWS UPD. A blue line drawn from the midpoint between the two melt curves in the nonduplication control sample indicates that ∼52% of the signal comes from the paternal melting curve (∼86.2°C) and 48% of the signal comes from the maternal melting curve (∼81.7°C). (c) A quantitative HRM analysis of multiple nonduplication control samples is shown, and although some variation in profiles could be detected, there was a very tight range of relative signal intensity in 15 nonduplication control samples between 52% and 60% (blue arrows). The individual melting curves ranged between 80.5°C and 82.3°C (average 81.4°C) for the paternal allele and 85°C–87°C (average 86°C) for the maternal allele. These values essentially match the peak intensity melt temperatures observed in (a) for the control. HRM, high-resolution melting; PWS/AS, Prader-Willi/Angelman syndrome; UPD, uniparental disomy.
FIG. 2.
FIG. 2.
Quantitative identification of maternal and paternal 15q duplications. Arrow heads indicate relative signal intensity between the two melting curves. (a) Quantitative HRM determination of parental origin using normalized melt profiles. Bisulfite-treated DNA samples from 28 individuals with interstitial duplications of unknown origin were analyzed by quantitative HRM and compared to AS deletion (red) and PWS-UPD (dark green) controls. One nonduplicated control DNA sample is indicated in gray at ∼50% relative signal intensity. Normalized melting profiles revealed a clear grouping of 26 maternal duplication samples (blue) at an average relative signal intensity of ∼66% and a grouping of 2 paternal duplication samples (green) at a relative signal intensity of ∼40%. (b) Detection of parental origin in stable inherited interstitial 15q duplications. HRM normalization analysis in a family, where the child (blue = 66% relative signal intensity) has inherited a maternal duplication and his mother (green = 39% relative signal intensity) a duplication of paternal origin. As expected, the father, who does not have a chromosomal duplication, showed a relative signal intensity of 52%, within the 52%–60% range found in normal controls. (c) Quantitative HRM can determine both parental origin and distinguish between interstitial and isodicentric duplications on 15q. The colors indicate HRM analysis in AS deletion (red), PWS UPD (dark green), nonduplication control samples (gray), and maternal interstitial duplication (blue). The two known isodicentric duplication samples are indicated in pink. One sample had an HRM relative signal intensity of 73%, indicating a 2.7:1 ratio of maternal to paternal signal, while the other had a signal intensity of 87%, consistent with a 6.7:1 maternal-to-paternal ratio. Both isodicentric 15q duplication samples showed relative signal intensities >66%, the observed interstitial duplication relative signal intensity.

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