Detection of an alternatively spliced variant in the PTPN22 gene

Richard Wagner


Autoimmune diseases are caused by the loss of self-tolerance and expansion of self-reactive lymphocytes that lead to tissue damage. Infectious agents may trigger some autoimmune disorders that express epitopes resembling self-antigens and that are led to sensitization of the individual against that tissue. However many autoimmune disorders are caused by internal dysregulation of the immune system without the apparent participation of infectious agents (Murphy, 2012).

Autoimmune diseases are a clinically diverse group of complex diseases. The protein tyrosine phosphatase N22 (PTPN22) gene exhibits regulatory activities for both T cells and B cells. PTPN22 encodes lymphoid phosphatase (Lyp), an intracellular tyrosine phosphatase (Cohenet al., 1999). Exclusively haematopoietic cells express Lyp. A missense single-nucleotide polymorphism (SNP) within this gene (1858C>T, rs2476601) has been associated with autoimmune diseases.

The Arg620Trp locus within the PTPN22 gene has been reported to increase autoimmune susceptibility associated with: Type 1 diabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Hashimotos thyroiditis, Graves disease, Addisons disease, Myasthenia Gravis, Vitiligo, Systemic sclerosis, and juvenile idiopathic arthritis

Allelic variants in the PTPN22 gene could predispose an individual to a more general autoimmune diathesis. The PTPN22 locus is one of the strongest risk factors outside of the major histocompatibility (MHC) complex that associates with autoimmune diseases. (Stanford & Bottini, 2014).

This study showed the expression of an alternatively spliced variant of the PTPN22 transcript in HL60 cells that introduces 28 new amino acids in a region in close proximity to a SNP (Arg620Trp).

Materials and Methods

RNA from HL60 myeloid cells was isolated. RT-PCR was performed using a variety of primer pairs spanning the 21 exons.

Primers were chosen thought the development of an intelligent software algorithm. This technology is fundamentally different than other technologies as it employs a weighted neural net to optimized primer selection.

RT-PCR was performed. The amplified products were run on a 2% agarose gel and visualized using a BioRad UV Transilluminator. PCR products were purified. The purified PCR product was sequenced. The sequenced information was then used to perform a BLAST analysis.


The primer pair for exon 15-17 produced two bands 209 and 125 base pairs in length. The results of the BLAST search showed that the larger, 209bp transcript variant included 84bp of intron 15 not present in the smaller, 125bp transcript variant.


The results presented reveal the presence in HL60 cells of an alternatively spliced PTPN22 transcript, which when translated generates a protein containing a 28 amino acid insertion that is located in close proximity to the Arg620Trp SNP, which has been associated with autoimmune disease. The biological activity of the splice variant observed in this investigation is not known. The close proximity of this alternative splicing event to the Arg620Trp SNP is a compelling motivation for further investigation.


PTPN22 encodes a tyrosine phosphatase that is expressed by haematopoietic cells. It regulates immune homeostasis by inhibiting T cell receptor signaling. In addition, it selectively promotes type I interferon responses following activation of myeloid-cell pattern-recognition receptors. PTPN22 contains three domains: N‑terminal PTP catalytic domain, an interdomain region, and a C‑terminal domain. The C‑terminal domain consists of four proline-rich regions that function as motifs for interaction with other proteins. A single nucleotide polymorphism (SNP) in the C‑terminal domain of PTPN22 disrupts an interaction motif in the protein. This study showed the expression of an alternatively spliced variant of the PTPN22 transcript in HL60 cells which introduces 28 new amino acids in a region in close proximity to a SNP (Arg620Trp) which has been associated with autoimmune diseases. Visualization of this region using predictive protein folding technology was performed to help elucidate the potential effects of this insertion on the biological activity of the protein.


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Figure 1-Detection of two alternatively spliced transcript variants between exons 15-17.

Figure 2-Diagram of the two alternatively spliced transcript variants observed in this study.

Figure 3-Sequences at intron-exon boundaries.

Figure 4-Exon 15 transcript with the inclusion of 84bp from intron 15 shown in red.

Understanding the effect of this alternative splicing event on the function of PTPN22 in immune-cell homeostasis requires more molecular research. In the innate immune system Lyp selectively regulates type 1 interferon (IFN) production after toll-like receptors (TLR) engagement in myeloid cells. TRLs are innate receptors on macrophages, dendritic cells, and some other cells, that recognize pathogens and their products, such as bacterial lipopolysaccharide (LPS). Recognition stimulates the receptor-bearing cells to produce cytokines that help initiate immune responses. TLR triggers host defense responses including type 1 IFNs secretion. Lyp connects TLR signaling in innate immune cells with the induction of type 1 IFN-driven host defense and immunoregulatory responses (Wang, Y. et al., 2013).

To characterize the biological activity of the splice variant observed in this investigation IFN-β production following stimulation of marrow-derived macrophages (BMM) with LPS or the TLR agonist polyinosinic-polycytidylic acid (poly(I:C)) could be utilized to quantify effects of treatments that result in increased expression of the PTPN22 SNP (Arg620Trp) transcript. Differences in IFN-β production associated with alternatively spliced PTPN22 exon 15 would demonstrate the biological activity of the 28 amino acid insert.

Full Paper


I would like to thank Dr. Sylvia Anderson for her generous donation of RNA. I also want to acknowledge the efforts of Kate Reid and Catharina Grubaugh for their assistance and guidance during this project. I also want to extend my gratitude to Dr. Berish Rubin for his patience, support and mentorship.

This document was last modified 05/07/2015.
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