Analysis of the Expression and Single-Nucleotide Variant Frequencies of the Butyrophilin-like 2 Gene in Patients With Uveal Melanoma

Abstract

IMPORTANCE Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 (BTNL2; OMIM, 606000), which is associated with prostate cancer risk and autoimmune diseases. OBJECTIVE To investigate the expression and variant allele frequencies of BTNL2, a candidate gene for chromosome 6 amplification, in patients with UM. DESIGN, SETTING, AND PARTICIPANTS In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variants rs28362679 and rs41441651 with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1-and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015. MAIN OUTCOMES AND MEASURES Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival. RESULTS We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of the rs28362679 variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). The rs41441651 variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival. CONCLUSIONS AND RELEVANCE Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control...

Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1

“…Mf differentiation was confirmed by positivity for the pan-Mf marker CD68 ( 47 ), which ranged between 97 and 100% among different donors. Mf polarization was obtained by removing conditioned medium (CM) and then culturing M0 Mf for additional 24 h with fresh medium supplemented with 5% FCS and containing 100 ng/mL Ultra-Pure LPS from Escherichia coli O111:B4 (Sigma-Aldrich) (for M1 polarization) or 20 ng/mL hIL-4 (Miltenyi Biotec) (for M2 polarization), as previously described ( 48 , 49 ), under normoxic or hypoxic conditions. M0 Mf cultured with fresh medium without polarization factors were used as baseline control.…”

The biology of uveal melanoma

“…Several groups have assembled significant numbers of expression profiles of UM samples [182, 183, 221–223] which, in part, are publicly available. The TCGA dataset also contains gene expression profiles (GEPs) obtained by RNA sequencing.…”

“…We have recently reported expression of the B7 family member Butyrophilin-Like 2 (BTNL2) gene by UM cells and macrophages that might contribute to the definition of the specific immunologic niche [223]. …”

“…Indeed, depletion of macrophages strikingly inhibited intraocular melanoma growth in a syngeneic model [264] and, more recently, in a transgenic model of UM [266]. Expression of M1/M2 polarization genes is strongly associated with metastatic risk [223]. …”

Prediction of Disordered Regions and Their Roles in the Anti-Pathogenic and Immunomodulatory Functions of Butyrophilins

“…Broad functionality of the members of BTN family that ranges from the crucial roles in the formation, secretion, and stabilization of milk fat globules to regulation of the immune response [ 5 ], association of these proteins with various human pathologies, such as autoimmune diseases [ 75 , 76 ], cardiovascular disease [ 77 , 78 , 79 ], Kawasaki disease [ 80 ], diabetes [ 76 , 81 ], sarcoidosis [ 82 , 83 , 84 ], multiple sclerosis [ 85 , 86 ], and cancer [ 87 , 88 , 89 , 90 , 91 , 92 ], to name a few, and their ability to protect infants against various human pathogens [ 93 ] clearly put butyrophilins to the category of moonlighting proteins. The capability of a protein to have multiple unrelated biological functions (i.e., to be a moonlighting protein) is commonly associated with the presence of intrinsically disordered protein regions (IDPRs) [ 37 , 39 , 49 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 ].…”