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Letters to the Editor
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Figure 2. Quantification of the vari- ant allele ratio of blood-derived DNA using digital PCR. (A) Sequences of the primers and probes. (B) Blue indicates FAM fluorescence for the target variant allele; red indicates VIC fluorescence for the normal allele; green indicates FAM+VIC; and yellow indicates no amplification.
extracted from peripheral blood leukocytes using a GEN- ERATION Capture Column kit (QIAGEN, Tokyo, Japan). All exons and the exon-intron boundaries of PROS1 were polymerase chain reaction (PCR)-amplified using GoTaq G2 Hot Start Master Mixes (Promega, Tokyo, Japan). PCR products were sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Tokyo, Japan) and an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific). Sanger sequencing indicated a novel heterozygous nonsense variant in exon 3 (NM_000313.4: c.246T>G; p.Tyr82*) in the proband and her sister. However, neither parent showed this variant by Sanger sequencing (Figure 1B). Since the probability of the same de novo variant occur- ring in the proband and her sister was considered virtual- ly zero, this case was attributed to another cause such as the parents’ genetic mosaicism. We therefore attempted to quantify the variant allele ratio. First, digital PCR (dPCR) was performed using QuantStudio 3D Digital PCR Master Mix v2 (Thermo Fisher Scientific) and the QuantStudio 3D Digital PCR system (Thermo Fisher Scientific) (Figure 2). The variant allele ratio was 48.5% for the proband, 52.9% for the sister, 15.2% for the
father, and 0.1% for the mother (Figure 2B).
Second, we quantified the variant allele ratios using pyrosequencing with PyroMark Q24 (QIAGEN) to con- firm the reproducibility of paternal mosaicism using a dif- ferent principle of measurement (Figure 3). The results of pyrosequencing indicated that the variant allele ratios were 50% for the proband, 49% for the sister, 15% for the father, and 1% for the mother (Figure 3B). The vari- ant allele ratios were consistent between dPCR and pyrosequencing for all family members, suggesting the presence of somatic mosaicism in the father. Based on this result, a careful review of the Sanger sequencing data of the father revealed the presence of a very small G peak
with a normal T peak (Figure 1B).
Third, we evaluated the ratio of variant alleles in sali-
va-derived DNA to confirm the reproducibility of the somatic mosaicism presented in the blood-derived DNA of the father. The saliva-derived DNA was extracted and purified using Oragene Purifier (Kyodo International). The results of pyrosequencing showed that the variant allele ratios of the saliva-derived DNA were 50% for the proband, 50% for the sister, 17% for the father, and 2% for the mother (Figure 3C), confirming the reproducibility
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