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Letters to the Editor
of the father’s mosaicism. Generally, the detection limits of dPCR and pyrosequencing are said to be more sensi- tive than that of Sanger sequencing: Sanger sequencing 15-20%;8,9 pyrosequencing 5%;8,10 dPCR 0.1-1%.11,12 We performed pyrosequencing of five control subjects and an average variant allele ratio was 1% (data not shown). In addition, the detection limit of our pyrosequencing assay system calculated at 3d was 2% (data not shown). However, we could not examine the control subjects with our dPCR assay system. Based on the above, the variant allele ratios detected in the mother, 0.1% for dPCR and 1-2% for pyrosequencing, were below the
A
detection limits of their respective assay system and were, therefore, considered to be background. In this case, the paternal mosaicism was observed in the blood cells (mesoderm origin) and salivary gland cells (ecto- derm origin). This may suggest that the genetic variant might have occurred early in the fetal development process. However, the variant allele ratio of the two types of DNA collected in this study happened to be about 15%-17%, but the variant allele ratios in other organs have not yet been verified; this makes it difficult to determine when the variant occurred from the variant allele ratios. It is also assumed that the father's germ cells
BC
Figure 3. Quantification of variant allele ratios in blood- and saliva-derived DNA using pyrosequencing. (A) Sequences of the primers. The results of variant allele ratios quantified by pyrosequencing of blood-derived DNA (B) and saliva-derived DNA (C) from the proband and family members. The gray area shows the allele ratio of A and C at the variant locus. Since pyrosequencing was performed using reverse primers, the allele ratio of C represents the variant allele ratio.
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haematologica | 2022; 107(1)