case_images: PMC5760075_01
case_images: A&B - Case 1. Abnormal promyelocytes with rounded nuclei and dense cytoplasmic granularity and hypolobated neutrophils. C&D - Case 2. Relatively scanty cytoplasmic granularity of the promyelocytes. E&F - Case 5. Hypolobulated and hypogranular myeloid cells along with abnormal promyelocytes.
case_images: mjhid-10-1-e2018002f2.jpg
case_images: PMC5760075_05_mjhid-10-1-e2018002f2.jpg
case_images: f2-mjhid-10-1-e2018002
case_images: In addition, maturing myeloid cells, hypogranular and hypolobated neutrophils were also present (Figure 2, E & F).
case_images: PMC5760075_02
case_images: A&B - Case 1. Abnormal promyelocytes with rounded nuclei and dense cytoplasmic granularity and hypolobated neutrophils. C&D - Case 2. Relatively scanty cytoplasmic granularity of the promyelocytes. E&F - Case 5. Hypolobulated and hypogranular myeloid cells along with abnormal promyelocytes.
case_images: mjhid-10-1-e2018002f2.jpg
case_images: PMC5760075_05_mjhid-10-1-e2018002f2.jpg
case_images: f2-mjhid-10-1-e2018002
case_images: In addition, maturing myeloid cells, hypogranular and hypolobated neutrophils were also present (Figure 2, E & F).
case_images: PMC5760075_03
case_images: PMC5760075_04
case_images: PMC5760075_05
case_images: A&B - Case 1. Abnormal promyelocytes with rounded nuclei and dense cytoplasmic granularity and hypolobated neutrophils. C&D - Case 2. Relatively scanty cytoplasmic granularity of the promyelocytes. E&F - Case 5. Hypolobulated and hypogranular myeloid cells along with abnormal promyelocytes.
case_images: mjhid-10-1-e2018002f2.jpg
case_images: PMC5760075_05_mjhid-10-1-e2018002f2.jpg
case_images: f2-mjhid-10-1-e2018002
case_images: In addition, maturing myeloid cells, hypogranular and hypolobated neutrophils were also present (Figure 2, E & F).
case_images: PMC5760075_06
case_images: Cellular Morphology and PML-RARA fusion transcript detection. Abnormal promyelocytes with characteristic bilobed nuclei (B) and presence of abundant cytoplasmic granules which showed strong cytochemical myeloperoxidase positivity (D) is seen in the uppermost panel. Some unusual myeloid differentiation is also seen (A & C). PCR amplicon size (480 bp) of novel Bcr3 PML-RARA transcript on agarose gel (middle panel) and capillary (lower panel) electrophoresis.
case_images: mjhid-10-1-e2018002f3.jpg
case_images: PMC5760075_06_mjhid-10-1-e2018002f3.jpg
case_images: f3-mjhid-10-1-e2018002
case_images: Peripheral smear revealed 29% abnormal promyelocytes with characteristic morphology and presence of differentiating myeloid series cells (Figure 3). Cloning of the PCR product into a vector and Sanger sequencing confirmed the above sequencing results (Figure 3).
case_images: Characterization of novel Bcr3 PML-RARA variant by Sanger sequencing (Case 6). Schematic representation of novel PML-RARA variant transcript is shown in the upper panel. PML gene exons are shown the squares and RARA gene exons are shown in the circles. Insertion of 40 nucleotides from RARA intron2 is shown as a shaded black circle. Exact patient sequences are shown in dashed box below. The cDNA and gDNA (lower panels) from the patient were sequenced by Sanger sequencing and aligned to the exonic and/or intronic sequences of PML and RARA genes.
case_images: mjhid-10-1-e2018002f4.jpg
case_images: PMC5760075_06_mjhid-10-1-e2018002f4.jpg
case_images: f4-mjhid-10-1-e2018002
case_images: Agarose gel electrophoresis for PML-RARA transcript identification revealed a PCR amplicon size of approximately 480bp, which was larger than the expected Bcr3 fusion transcript size (~370bp) (Figure 4). The PCR amplicons were further subjected to Sanger sequencing and revealed an atypical, novel fusion pattern between exon4 of PML and exon 3 of RARA genes (Figure 4). Interestingly, an additional 40 nucleotides, mapped to intron 2 of RARA, were also coded as part of the mRNA fusion transcript (Figure 4). Long-range genomic DNA PCR followed by Sanger sequencing identified a breakpoint immediately downstream to exon 4 of PML gene (PML nucleotide no: 74024927) and distal to the 11,789th base (RARA nucleotide no: 40343186) within intron 2 of RARA gene (Figure 4).
case_images: PMC5760075_07
case_images: Characterization of Bcr 2 PML-RARA variant (Case 7). Agarose gel electrophoresis (upper panel) of our case (Case 7) along with known positive control (PC), biologically negative control (BNC) and a non-template control (NTC). Sanger sequencing (lower panel) of the same case shows fusion of PML exon 6 with RARA exon 3 and addition of 126 nucleotides from RARA intron 2.
case_images: mjhid-10-1-e2018002f5.jpg
case_images: PMC5760075_07_mjhid-10-1-e2018002f5.jpg
case_images: f5-mjhid-10-1-e2018002
case_images: Sanger sequencing revealed breakpoints within exon 6 of PML and exon 3 of RARA (RARA nucleotide no: 40351910) with the addition of 126 nucleotides, which mapped to the central portion of RARA intron 2 (Figure 5).