A common 56-kilobase deletion in a primate-specific segmental duplication creates a novel butyrophilin-like protein

Abstract

Background: The Butyrophilin-like (BTNL) proteins are likely to play an important role in inflammation and immune response. Like the B7 protein family, many human and murine BTNL members have been shown to control T lymphocytes response, and polymorphisms in human BTNL2 have been linked to several inflammatory diseases, such as pulmonary sarcoidosis, inflammatory bowel disease and neonatal lupus.

Results: In this study we provide a comprehensive population, genomic and transcriptomic analysis of a 56-kb deletion copy number variant (CNV), located within two segmental duplications of two genes belonging to the BTNL family, namely BTNL8 and BTNL3. We confirm the presence of a novel BTNL8*3 fusion-protein product, and show an influence of the deletion variant on the expression level of several genes involved in immune function, including BTNL9, another member of the same family. Moreover, by genotyping HapMap and human diversity panel (HGDP) samples, we demonstrate a clear difference in the stratification of the BTNL8_BTNL3-del allele frequency between major continental human populations.

Conclusion: Despite tremendous progress in the field of structural variation, rather few CNVs have been functionally characterized so far. Here, we show clear functional consequences of a new deletion CNV (BTNL8_BTNL3-del) with potentially important implication in the human immune system and in inflammatory and proliferative disorders. In addition, the marked population differences found of BTNL8_BTNL3-del frequencies suggest that this deletion CNV might have evolved under positive selection due to environmental conditions in some populations, with potential phenotypic consequences.

Figure 1

Region of the BTNL gene cluster containing the BTNL8_BTNL3-del allele and sequence structure of deletion. (A) The figure shows 200-kb of the genomic region on human chromosome 5. The expected transcripts of the three genes in this region are shown with their transcript orientation. The CNV region, which is indicated by the dashed line, removes 56-kb, from intron four of BTNL8 to intron four of BTNL3. The black-yellow dashed box represents the SD shared between BTNL8 and BTNL3. PCR assays were designed to distinguish between non-deleted and deleted alleles (short arrows show position of PCR oligonucleotides). The PCR assay amplifies a ~420-bp product for the non-deleted allele, and a ~340-bp fragment for the deleted allele (bottom). Results from the PCR assay are shown for seven LCLs. (B) The deleted allele shows long homologous stretches at its breakpoints (~1.6-kb sequence blocks of 98% identity, of which 300-bp are shown aligned here) and thus probably results from non-allelic homologous recombination (NAHR). The “cross-over” occurred somewhere within 112-bp of identical sequence indicated as “NAHR-region”.

Figure 2

World-wide distribution of the BTNL8_BTNL3-del allele in different human populations. Genotype analysis of populations is shown over a map of the world. The deletion frequency is indicated by the black part of the pie in the chart. Total number of individuals genotyped is given in red.

Figure 3

Identification of a chimeric BTNL8*3 product. (A) In LCLs carrying one or two copies of the BTNL8_BTNL3-del allele, a fusion BTNL8*3 mRNA product could be detected by RT-PCR. (B) Allelic expression differences are shown in individual tissues. In tissues heterozygous for the BTNL8*3 allele, the non-deleted BTNL8 allele was expressed threefold higher than the BTNL8*3 allele (all tissues combined). (C) Representative western blot analysis revealed a novel protein in LCL cells expressing the deleted allele. The band intensities were normalized to tubulin expression. (D) The putative protein resulting from the BTNL8*3 deletion CNV would contain the extracellular IgV-like and IgC-like domains and the TM domain of BTNL8 and the cytoplasmic B30.2 domain of BTNL3.

Figure 4

Effect of BTNL8-BTNL3_del on BTNL9 level in LCLs. BTNL9 mRNA and protein levels were measured by RT-qPCR and western-blot analysis, respectively. (A) For each genotype expression-level of five different samples were measured. Samples containing at least one deleted allele show significant reduced amount in BTNL9 mRNA level. (B) Representative western-blot shows expression of BTNL9 in cell homozygous for the deletion versus cells homozygous for the non-deleted allele. The band intensities were normalized to tubulin expression.

Figure 5

Differential expression of genes depending on BTNL8/BTNL3 genotype. (A) Nine genes could be confirmed by RT-qPCR analysis to be differentially expressed in LCLs homozygous for the BTNL8_BTNL3-del allele. Red, increased mRNA level; green, decreased mRNA level in LCLs homozygous for the CNV. (B) Network of genes formed by Ingenuity Pathway Analysis. Genes depicted in red (increased mRNA level) and green (decreased mRNA level) were confirmed by RT-qPCR analysis to be differentially expressed in LCLs homozygous for the CNV. Genes depicted in white were not found to be deregulated even though they are part of the same network.