Assessment of a high-throughput DNA melting analysis assay for rapid screening of gene variants in the ornithine transcarbamylase gene

K. Sumner*, L. Hubley*, S. Dobrowolski*, G. Pont-Kingdon*, R. Margraf*, H. Best*†, E. Lyon*†ARUP Institute for Clinical and Experimental Pathology *, Salt Lake City, UT, Department of Pathology †,

University of Utah, Salt Lake City. kelli.sumner@aruplab.com

Ornithine transcarbamylase (OTC) deficiency is the most

common inherited defect of the urea cycle and is caused by

mutations in the OTC gene (Xp21.1). Over 450 disease-causing

mutations have been reported, most of which are family

specific, with the vast majority occurring within the exons and

intron/exon boundaries of the OTC gene. All 10 exons in the OTC

gene are smaller than 155 base pairs making this an ideal target

for exon scanning.

Exons and intron/exon boundaries were amplified in the

LightCycler 480 Real-Time PCR System (Roche Applied Science,

Indianapolis, IN) using the LightScanner Master Mix with

LCGreen Plus+ Melting Dye (Idaho Technology Inc., Salt Lake City,

UT) and subsequently melted on the same instrument or

amplified on the GeneAmp PCR System 9700 (Applied

Biosystems, Carlsbad, CA) and melted on the LightScanner

System (Idaho Technology, Inc., Salt Lake City, UT). Exon

scanning primers were taken from Dobrowolski et al, and M13

tails were added for sequencing.

Melting curves were analyzed for all ten exons. A second

analysis was done using a small amplicon genotyping melt to

further characterize deviant profiles in exons that contain

common OTC polymorphisms (exons 2, 4 and 8). Melting

profiles were obtained for 11 de-identified, normal female

samples submitted to ARUP Laboratories for unrelated testing.

The OTC gene was sequenced in each of these patients to

confirm melting curve results.

A second study was done using 3 positive samples purchased

from Coriell Institute for Medical Research (Camden, New

Jersey), NA06902, NA09039, and NA 09040 with confirmed

diagnosis of OTC deficiency. Male samples were spiked using

genomic DNA from a known normal sample prior to PCR

amplification in order to obtain heteroduplex melts. Samples

were amplified as before and melting curves were then analyzed

for difference in melting signatures from the normal control.

Ninety-nine out of the 100 exons analyzed were concordant with their

sequencing results. Therefore, the assay had an overall analytical

sensitivity (TP/TP+FN) of 100% and an analytical specificity (TN/TN+FN)

of 99% (excluding all exons from the poorly amplified sample) when

amplified on the LightCycler 480 using the LightScanner Master Mix

with LCGreen Plus+ dye and melted on the same instrument. Positive

samples were identified from the normal samples by deviant melting

profiles. In summary, DNA Melting analysis is a rapid and inexpensive

method to screen for OTC deficiency.

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Yamaguchi et al. Mutations and Polymorphisms in the human ornithine transcarbamylase (OTC) gene,

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No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

Two samples had deviant melting curves and were confirmed to be c.137A>G heterozygous (p.K46R) by sequencing. One sample that was

grouped with the normal samples was confirmed to be c.137A>G homozygous (p.K46R).

No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

Three samples had deviant melting curves and were confirmed to be c.299-8T>A

heterozygous by sequencing.

No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

No sample had a deviant melting curve and all were confirmed to be normal by

sequencing.

Two samples had deviant melting curves and were confirmed to contain variants.

Both contained the variant c.809A>G (p.Q270R) and one had an additional

variant c.867+35T>G

Redundant Genotyping Melt

Positives

The sample containing homozygous c.137A>G (p.K46R) was not distinguishable from normal

when using the LightCycler480 instrument.

The sample containing homozygous c.137A>G (p.K46R)was distinguishable

from normal samples when amplified on the GeneAmp PCR System 9700 and

melted on the LightScanner System and LightScanner Software for analysis.

The samples containing heterozygous c.299-8T>A were distinguishable from

wild-type samples when using the LightCycler 480 instrument.

The samples containing heterozygous c.809A>G were distinguishable from normal

samples when using the LightCycler 480 instrument.

The sample containing c.137A>G (p.L46R) was

distinguishable from normal samples and from the

positive, c.140A>T (p.N47I) sample.

The positive sample containing c.422G>A

(p.R141Q) was distinguishable from normal

samples.Overall results for positive samples. Male samples were mixed with a known normal female sample. There was a 100% correlation between scanning

results and sequencing results

One sample (A) was excluded from all analysis due to poor

amplification. Ninety-nine out of 100 exons analyzed were

concordant with their sequencing results. Seven exons from 3

samples had deviant melting curves, sequencing confirmed

benign variants. One sample with a homozygous polymorphism

had a melt similar to normal samples when using the LightCycler

480 but was distinguishable from the normal samples using the

genotyping melt assay when the sample was amplified on the

GeneAmp PCR System 9700 and melted on the LightScanner

System.

The 3 Coriell samples had deviant melts in exons 2 and 5, and

results were confirmed by sequencing. One sample had deviant

melting curves in both exons (2 and 5); sequencing confirmed

the presence of the c.137A>G (p.L46R) benign variant and a

c.422G>A (p.R141Q) pathogenic variant. Another sample with a

deviant melting curve in exon 2 was confirmed to contain a

c.140A>T (p.N47I) pathogenic variant. The 3rd sample had a

deviant melting curve in exon 5 and was found to contain a

c.422G>A (p.R141Q) pathogenic variant.

Abstract

Conclusions References

Results

Materials and Methods

Exon Scanning