Identification and interpretation of TET2 noncanonical splicing site intronic variants in myeloid neoplasm patients

Abstract Background: DNA hypermethylation and instability due to inactivation mutations in Ten–eleven translocation 2 (TET2) is a key biomarker of hematological malignancies. This study aims at characterizing two intronic noncanonical splice‐site variants, c.3954+5_3954+8delGTTT and c.3954+5G>A. Methods: We used in silico prediction tools, reverse transcription (RT)‐PCR, and Sanger sequencing on blood/bone marrow‐derived RNA specimens to determine the aberrant splicing. Results: In silico prediction of both variants exhibited reduced splicing strength at the TET2 intron 7 splicing donor site. RT‐PCR and Sanger sequencing identified a 62‐bp deletion at the exon 7, producing a frameshift mutation, p.Cys1298*. Conclusion: This study provides functional evidence for two intronic TET2 variants that cause alternative splicing and frameshift mutation.


Patient samples, DNA and RNA extraction, next-generation sequencing
Genomic DNA and total RNA were extracted from either bone marrow or peripheral blood of the same specimens. A nonleukemic patient was used as the negative control. Two TET2 intronic variants c.3954+5_3954+8delGTTT (present in one patient) and c.3954+5G>A (present in two patients) were identified from a F I G U R E 2 (A) Graphic representation showing TET2 splicing products with or without TET2 intronic variants (c.3954+5_3954+8delGTTT, c.3954+5G>A). Breakpoints at the cryptic and original 5′ splice site junctions are shown with blue triangles. The primer binding sites and subsequent PCR products in WT and mutant TET2 spliced mRNA are also shown. (B) RT-PCR analysis on RNA from a negative control patient and a patient with TET2 c.3954+5_3954+8del variant. Gel electrophoresis image showing PCR products amplified using specific primers binding to Ex6 (forward) and Ex8 (reverse). Fragment size of 246 bp indicates TET2 wild-type sequences (ex6+ex7+ex8) and fragment of 185 bp size (ex6+ partial ex7+ex8) indicates the TET2 splicing variant. Only wild-type product was identified in the negative (WT) control. No visible product is shown in no template control (NTC).

Identification of two intronic TET2 variants in hematoneoplasm patients
Two novel TET2 (NM_001127208.2) intron 7 variants with high VAFs were identified in three patients (Table 1)

Confirmation of impact in splicing in two novel TET2 variants
The two intronic variants' impact on TET2 gene splicing was further characterized by RT-PCR and Sanger sequencing at the RNA level. As shown in Figure 2A,B, a band at approximately 246 bp region corresponding to the wild-type sequence was seen in the negative (WT) con-trol. However, for the patient with deletion mutation (patient 1), two bands were seen; one was at 246 bp, and the other appeared just below 200 bp. This ∼200 bp RT-PCR product was also obtained for patients 2 and 3 (data not shown) along with 246 bp WT product. Sanger sequencing of the extracted 246 bp band from the WT control consists of all expected exonic sequences, including partial exon 6, full exon 7, and partial exon 8. Interestingly, sequencing of the ∼200 bp products from both cases of c.3954+5G>A and c.3954+5_3954+8delGTTT variants, as shown in Figure 3, revealed a 62-bp deletion at the 3′ portion of the exon 7. This deletion used an exon 7 internal cryptic splicing site c.3892 (g.106180864) to produce an alternative splicing product that is predicted to cause a protein reading frameshift mutation, p.Cys1298*.
These results demonstrate that both variants lead to the same premature termination codon that is expected to result in a truncating protein. The activation of 5′ cryptic or generation of de novo splice sites is mostly due to alteration in the first intron nucleotide (+1) or +5 position [9]. Position +5 in the intron is required for base pairing with U1 and U6 snRNA components of spliceosomes during the splicing process [10]. Since both variants, c.3954+5_3954+8delGTTT and c.3954+5G>A, in our study encompass the +5G intron position, our finding of the splicing product with a 62-bp deletion of exon 7 corroborates with our understanding of pre-mRNA splicing mechanism and demonstrates the usefulness of in silico prediction tools.

ACKNOWLEDGMENTS
This study was in part supported by the research fund of the Laboratory Medicine Department of the Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic.

CONFLICT OF INTEREST STATEMENT
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

DATA AVAILABILITY STATEMENT
Data are available upon request due to privacy/ethical restrictions.

NUMBER)
This study has not been registered for any clinical trial.

PATIENT CONSENT STATEMENT
n/a

ETHICS STATEMENT
The study was conducted according to the approved protocols of Cleveland Clinic's Institutional Review Board (IRB; 17-177 and 19-329).