Heightened BRAF and BRAF pseudogene expression levels in 2 Japanese patients with Erdheim‐Chester disease

Erdheim‐Chester disease (ECD) is a rare form of non‐Langerhans cell histiocytosis with multi‐organ involvement. Many cases have mutations in the BRAF and other genes involved in the MAPK activation pathway. Pseudogenes, which regulate their parental genes post‐transcriptionally, are overexpressed in various tumors, but we found no previous reports of high pseudogene expression in ECD.

ECD, but only 50% of patients describe bone pain. Other hallmark manifestations are periaortic sheathing ("coated aorta"); pericarditis; myocardial infiltration; pulmonary interlobular septal thickening; perinephric infiltrates ("hairy kidneys"); central nervous system involvement; orbital infiltration, resulting in exophthalmos; endocrinopathies, such as diabetes insipidus; and skin xanthomas and xanthelasmas. It is difficult to diagnose ECD without cutaneous findings in early-stage disease because symptoms are often nonspecific and subjective. Sometimes ECD is only diagnosed at autopsy. [3][4][5][6] Dermatologic findings of ECD include xanthomas of the face, neck, axilla, and trunk, which are found in approximately one-third of people with ECD. 7 Xanthelasmas appear in approximately 25% of patients with ECD. 8 The presence of skin manifestations leads to earlier diagnosis because the lesions are easily identified by dermatologists, and it is easier to obtain a biopsy from the skin than from other organs.

| Reverse transcription PCR and real-time PCR
Total RNA was isolated using Maxwell 16 LEV simplyRNA Tissue Kit.
RNA concentration was measured with the NanoDrop 2000 spectrophotometer, and RNA integrity was verified on 2% denaturing agarose gels.
ReverTra Ace qPCR RT Master Mix (TOYOBO) was used for RNA reverse transcription. The reaction was conducted in a 10 lL mixture.
Total RNA was reverse-transcribed, and real-time PCR was performed to amplify cDNA via primer sets specific for either BRAF or BRAF pseudogenes-speS, speAS, pS1, pS2, and pAS ( Figure 3,   Table 1) 17 was amplified in both patients. The BRAFV600E mutation was detected in ECD1, but not in ECD2, during sequence analysis (Figure 4).

BRAF and BRAF pseudogenes compared with normal tissues
No genomic contamination was evident, as no amplification was observed when using mRNA as a template. BRAF pseudogene expression by PC3 cells was approximately 1000 times lower than BRAF expression using the standard curve method. Both BP-0 and ECD and in the positive control (PC3 cells), but not in NH1 normal skin. BRAF genes were amplified in patients and control samples ( Figure 5). The PCR products using BP-0 primers were 449 base pairs (bp) and using BP-1 primers were 334 bp, including the intron.
Thus, there was no splicing out of the BRAF pseudogene, as previously reported, 16,17 and no mutation in the PCR products based on sequencing analyses. Expression levels of both BRAF and BRAF pseudogenes were higher in xanthomas than in normal tissues ( Figure 5).

| DISCUSSION
The macrophages in our ECD cases showed positivity of CD163, which shows M2-like polarized tumor-associated macrophages promoting cancer cell growth. 18