jnmaciuch
Senior Member (Voting Rights)
Figures for the different BTN2A1 isoforms and their tissue-specific transcript expression, as promised. As a reminder, most of the ME/CFS-associated mutations fell in the PRY-SPRY domain, which is only present in 2/4 isoforms (the two on the left in the figure).
I forgot I can't upload a markdown document, but here's the code:
Code:
---
title: "Tissue-specific expression analysis of BTN isoforms"
output: html_document
date: "2026-08-29"
---
```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)
# Set working directory
workDir <- "~/Documents/coding"
# Load packages
library(jsonlite)
library(dplyr)
library(ggplot2)
```
BTN2A1 has several isoforms with different protein domains. Per the [AstraZeneca](https://azphewas.com/QVView/6319c068-fd59-46d8-85ee-82d82482eb14/BTN2A1/8e436248-c6c7-4c3c-9520-6a03495b72af/f5e4d803-b578-4371-9c76-b43baf74d772/db9a8d3d-b11a-4b5d-a129-4327844bcfb1/binary/EUR) rare mutation analysis, most ME/CFS-associated mutations were in the cytosolic PRY-SPRY domain.
Per [UniProt](https://www.uniprot.org/uniprotkb/Q7KYR7/entry), we know of 4 protein isoforms of BTN2A1. From the "Genome annotation databases" section, I manually pulled the associated transcript IDs (e.g. ENST00000312541.10) for all listed isoforms (only the ones matching BTN2A1 GENCODE ID ENSG00000112763.18, since ENSG00000292170.1 is not used in GTEx).
Clicking on the transcript ID link takes you to the Ensembl entry, where I found information on whether the isoform contained an SPRY domain via the "Transcript function" button. I compiled this information manually into a spreadsheet.
```{r}
# Load table for Uniprot transcripts and isoforms of BTN2A1
BTN_df <- read.csv(file.path(workDir,
"BTN2A1_isoform_table.csv"))
```
To test the hypothesis that certain protein isoforms with or without this protein domain had different expression levels in the cerebellum and/or immune cells, I pulled the median transcript expression values from the GTEx API using the call:
```{bash}
curl -X 'GET' \
'https://gtexportal.org/api/v2/expression/medianTranscriptExpression?gencodeId=ENSG00000112763.17&datasetId=gtex_v10&tissueSiteDetailId=Adrenal_Gland&tissueSiteDetailId=Brain_Amygdala&tissueSiteDetailId=Brain_Anterior_cingulate_cortex_BA24&tissueSiteDetailId=Brain_Caudate_basal_ganglia&tissueSiteDetailId=Brain_Cerebellar_Hemisphere&tissueSiteDetailId=Brain_Cerebellum&tissueSiteDetailId=Brain_Cortex&tissueSiteDetailId=Brain_Frontal_Cortex_BA9&tissueSiteDetailId=Brain_Hippocampus&tissueSiteDetailId=Brain_Hypothalamus&tissueSiteDetailId=Brain_Nucleus_accumbens_basal_ganglia&tissueSiteDetailId=Brain_Putamen_basal_ganglia&tissueSiteDetailId=Brain_Spinal_cord_cervical_c-1&tissueSiteDetailId=Brain_Substantia_nigra&tissueSiteDetailId=Breast_Mammary_Tissue&tissueSiteDetailId=Cells_Cultured_fibroblasts&tissueSiteDetailId=Cells_EBV-transformed_lymphocytes&tissueSiteDetailId=Colon_Sigmoid&tissueSiteDetailId=Colon_Transverse&tissueSiteDetailId=Liver&tissueSiteDetailId=Lung&tissueSiteDetailId=Minor_Salivary_Gland&tissueSiteDetailId=Muscle_Skeletal&tissueSiteDetailId=Nerve_Tibial&tissueSiteDetailId=Pancreas&tissueSiteDetailId=Pituitary&tissueSiteDetailId=Skin_Not_Sun_Exposed_Suprapubic&tissueSiteDetailId=Skin_Sun_Exposed_Lower_leg&tissueSiteDetailId=Small_Intestine_Terminal_Ileum&tissueSiteDetailId=Spleen&tissueSiteDetailId=Stomach&tissueSiteDetailId=Thyroid&tissueSiteDetailId=Whole_Blood&tissueSiteDetailId=Adipose_Subcutaneous&tissueSiteDetailId=Adipose_Visceral_Omentum&tissueSiteDetailId=Artery_Aorta&tissueSiteDetailId=Artery_Coronary&tissueSiteDetailId=Artery_Tibial&tissueSiteDetailId=Bladder&tissueSiteDetailId=Cervix_Ectocervix&tissueSiteDetailId=Cervix_Endocervix&tissueSiteDetailId=Esophagus_Gastroesophageal_Junction&tissueSiteDetailId=Esophagus_Mucosa&tissueSiteDetailId=Esophagus_Muscularis&tissueSiteDetailId=Fallopian_Tube&tissueSiteDetailId=Heart_Atrial_Appendage&tissueSiteDetailId=Heart_Left_Ventricle&tissueSiteDetailId=Kidney_Cortex&tissueSiteDetailId=Kidney_Medulla&tissueSiteDetailId=Ovary&tissueSiteDetailId=Prostate&tissueSiteDetailId=Testis&tissueSiteDetailId=Uterus&tissueSiteDetailId=Vagina&page=0&itemsPerPage=100000' \
-H 'accept: application/json'
```
```{r}
# Import transcript counts obtained from API call
counts <- fromJSON(file.path(workDir,
"response_1788031490143.json"))$data
```
Note that I am making an assumption that detection of the alternatively spliced transcript correlates to actual protein expression.
This call pulls all available transcript counts for the gene, so I subset to transcripts that had information in Uniprot:
```{r}
# Keep only counts for transcript IDs in table
counts <- inner_join(BTN_df,
counts,
by = "transcriptId")
```
And now I plot the results:
```{r}
# Some additional formatting for plotting:
counts <- counts %>%
mutate(SPRY_domain = if_else(SPRY_domain, "Contains SPRY domain", "No SPRY domain"),
SPRYId = paste(SPRY_domain, uniprotId, sep = " | "))
# Load color scheme for tissues (from https://github.com/broadinstitute/gtex-v8/blob/master/data/gtex_colors.txt)
color_df <- read.delim(file.path(workDir,
"gtex_colors.txt"))
# Create color scale vector
color_scale <- c(color_df$color_hex) %>% setNames(color_df$tissue_id)
# Plot
ggplot(counts,
aes(y = tissueSiteDetailId,
x = median)) +
geom_col(aes(fill = tissueSiteDetailId)) +
scale_fill_manual(values = color_scale) +
facet_wrap(vars(SPRYId, transcriptId), nrow = 1) +
theme_bw() +
theme(axis.text.x = element_text(angle = 90, hjust = 1)) +
guides(fill = "none")
```I plotted all available organs, specifically paying attention to the cerebellum and immune cell "tissues" (Whole Blood, Spleen, EBV-transformed lymphoblasts, cultured fibroblasts). While the two SPRY-containing isoforms (Q7KYR7-2 and Q7KYR7-5) had highest levels in the cerebellum, each was roughly matched by at least one immune tissue (fibroblasts and spleen, respectively). High expression in the arterial tissues matching cerebellar expression was also notable.
Overall, there is not a strong and clear indication that the BTN2A1 isoforms containing regions with ME/CFS-associated mutations are much more strongly expressed in brain vs. immune cells. Though analysis is limited by lack of resolution on specific immune cell types.
And the referenced files are attached
Last edited:
