The First Molecular Profiling of JAK2 Exon 12 and Flanking Intronic Sequences in Indonesia.

Abstract

Objective: Data on JAK2 Exon 12 mutations in Indonesia are not available. This study aimed to explore the presence of known JAK2 exon 12 coding mutations in V617F-negative myeloproliferative neoplasm (MPN) patients in Indonesia and to describe any novel intronic changes downstream of exon 12.

Methods: A total of 198 V617F-negative MPN samples were screened using allele-specific polymerase chain reaction (AS-PCR) for four commonly reported canonical exon 12 mutations. Of these, 57 cases were selected at random or for atypical PCR band patterns and underwent bidirectional Sanger sequencing of a 496-bp fragment covering exon 12 plus ~250 bp of the adjacent region. Variants were cross-referenced with gnomAD v4.1.0. Furthermore, SpliceAI was used to predict splice site gain or loss.

Results: No coding mutations were identified in exon 12. However, two intronic variants were detected adjacently downstream: NC_000009.12:g.5070231_5070235delTCTTA in 29 of 57 cases (50.9%), which was not present in the gnomAD database, and NC_000009.12:g.5070319C>T in 1 of 57 cases (1.8%), corresponding to a rare gnomAD entry. The SpliceAI Δ scores remained below 0.2, a high-sensitivity threshold, indicating no predicted impact on canonical splicing.

Conclusion: Classic exon 12 coding mutations are apparently absent in Indonesian V617F-negative MPNs. We report a highly frequent downstream intronic deletion not recorded in global databases, as well as a rare single nucleotide variant (SNV) that aligns with gnomAD data. Although in silico tools suggest minimal splice disruption, RNA-level assays are needed to rule out the subtle effects of the variant. Our workflow, which combines allele-specific PCR and targeted sequencing, may be suitable for laboratories facing resource constraints.