A pyrosequencing-based assay for the rapid detection of the 22q11.2 deletion in DNA from buccal and dried blood spot samples.
Author(s): Koontz D, Baecher K, Kobrynski L, Nikolova S, Gallagher M
Publication: J Mol Diagn, 2014, Vol. 16, Page 533-40
PubMed ID: 24973633 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of specimen type and analytical variables on the detection of the 22q11.2 deletion (diGeorge syndrome) by pyrosequencing.
Conclusion of Paper
Multiplex ligation-dependent probe amplification (MLPA) was able to amplify DNA from buccal swabs but not dried blood spots (DBS), however, the presence of the 22q11.2 sequence was confirmed in DBS using FISH or pyrosequencing. With chromosome 18 used as a reference, it was possible to determine 22q11.2 copy number by pyrosequencing using C/T peak height ratios in DBS and buccal cells, but it was harder to distinguish deletion of the sequence versus no deletion using A/G ratios. Optimal PCR annealing temperatures depended on the specimen type, but for all specimen types the PyroMark PCR kit produced the lowest coefficient of variance (CV) in pyrosequencing C/T ratios versus the AmpliTaq Gold kit, with or without a PCR boost. While copy number was successfully determined using 0.5 ng of DNA from all specimen types, for DBS, the groups were close together unless 2.0 ng were used.
Studies
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Study Purpose
The purpose of this study was to determine the effects of using DBS, buccal swabs or whole blood, amplification kit type, input amount, and analytical properties on the detection of the 22q11.2 deletion by pyrosequencing. The study included 19 paired blood specimens and buccal swabs, 9 non-paired blood specimens and 1 non-paired buccal swab specimen from patients with a 22q11.2 deletion (DiGeorge syndrome) and 180 blood specimens, 88 buccal swabs and 15 DBS from individuals without a 22q11.2 deletion. DNA was extracted from DBS using the Qiagen Investigator kit, from whole blood using the Gentra Puregene kit, and from buccal cells using phenol-chloroform. PCR conditions were tested using 147 blood specimens and 16 buccal swabs from patients without a 22q11.2 deletion and 19 blood specimens and 6 buccal swab specimens from individuals with the deletion. The effect of template amount was determined using 2 blood specimens, 3 buccal swabs and 3 DBS from individuals without the deletion and 3 buccal swabs and 2 DBS from individuals with a 22q11.2 deletion. The preservation method of the liquid blood and buccal cell specimens was not specified and assumed to be fresh.
Summary of Findings:
MLPA was able to amplify DNA from buccal swabs but not DBS. However, the presence of the 22q11.2 sequence was confirmed in 4 of 15 DBS using FISH and in the other 11 of 15 DBS using pyrosequencing. With chromosome 18 used as a reference, it was possible to determine 22q11.2 copy number by pyrosequencing using C/T peak height ratios in DBS and buccal cells. When the A/G peak height was used instead of C/T, it was harder to distinguish deleted versus non-deleted specimens, with only 98.9% specificity using liquid blood specimens. However, 100% sensitivity and specificity were still obtainable using A/G peak height for DBS and buccal cells. The annealing temperature that provided optimal separation of specimens based on C/T peak height ratios differed slightly between specimen types with a higher optimal temperature for DNA from DBS (63.5°C) than DNA from liquid blood or buccal cells (60.5°C). The CV of the pyrosequencing C/T ratios for non-deleted specimens was lower for liquid blood and buccal cell specimens (DBS not tested) when the PyroMark PCR kit was used rather than the AmpliTaq Gold kit (p<0.0001) or the AmplitTaq Gold plus PCR boost (p<0.0023). Importantly, the CV of non-deleted specimens was not significantly affected by PCR kit. While copy number was successfully determined using 0.5 ng of DNA from all specimen types, for DBS, the groups were close together unless 2.0 ng were used.
Biospecimens
Preservative Types
- None (Fresh)
- Other Preservative
Diagnoses:
- Other diagnoses
- Normal
Platform:
Analyte Technology Platform DNA DNA sequencing DNA PCR DNA FISH Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) DNA sequencing Specific Reaction solution PyroMark PCR kit
AmpliTaq Gold kit
AmpliTaq Gold kit plus PCR boost
Preaquisition Patient genotype 22q11.2 deletion
22q11.2 sequence present
Biospecimen Acquisition Biospecimen location DBS
Buccal cells
Biospecimen Preservation Type of fixation/preservation Air-dried
None (fresh)
DNA sequencing Specific Targeted nucleic acid 22q11.2
Chromosome 18
DNA sequencing Specific Template/input amount 0.125 ng
0.25 ng
0.5 ng
1.0 ng
2.0 ng
DNA sequencing Specific Data handling A/G peak height ratio
C/T peak height ratio
DNA sequencing Specific Incubation temperature 60.5°C
63.5°C