Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep

Abstract

In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.

Figure 1. Estimating the frequency of highly-susceptible TMEM154 alleles in global sheep populations.

The “c” allele of SNP OAR17_5388531 is in linkage disequilibrium with the “g” nucleotide allele in codon 35 (gaa) of TMEM154. Genotypes for OAR17_5388531 were derived from the ISGC ovine SNP50k data set . Numbers in parentheses for each breed group indicate the number of animals genotyped. The 11 breed groups with asterisks were genotyped for TMEM154 E35 by Sanger sequencing and were included for comparison with the 74 ISGC breed groups.

Figure 2. TMEM154 SNP maps and median-joining networks.

Panel A, genomic map of TMEM154: orange arrows, 5′ and 3′untranslated regions of exons; blue arrows, exon coding regions; grey rectangles, introns or intergenic regions. Blue and red tick dots denote position and frequency of SNPs in an international panel of 75 sheep and a panel of 96 U.S. sheep , respectively. Panel B, high resolution map of TMEM154 regions targeted for PCR-amplification. PCR amplification primers are indicated with black arrowheads and listed in Table S3. Red lowercase letters above SNP positions are IUPAC/IUBMB ambiguity codes for nucleotides (r = a/g, y = c/t, m = a/c, k = g/t, s = c/g, w = a/t) and indicate 12 sites affected by nonsynonymous substitutions. The red uppercase letters above SNP positions indicate the amino acid polymorphisms encoded at TMEM154 codons 4, 13, 14, 25, 31, 33, 35, 44, 70, 75, 82 and 102. Black lowercase letters below SNPs indicate nucleotide polymorphisms that resulted in synonymous substitutions. Panel C, the areas of circles for haplotypes 1 to 4 are proportional to the frequencies in the international panel of 75 ISGC sheep. The symbols are as follows: black circles, risk factors; white circle, non-risk factor; grey circles, risk factor status unknown; red circles, haplotypes known in U.S. sheep but not observed in the international panel of 75 ISGC sheep (risk factor status unknown); shaded square, TMEM154 haplotype predicted to have occurred but unobserved to date. Dashed grey line, haplotypes observed in wild sheep species but not domestic sheep. Haplotypes 13 and 14 were observed in one animal each and their location of origin is indicated.

Figure 3. Evidence for TMEM154 missense mutations in whole genome sequences data from an international panel of 75 sheep.

Computer screen images of Integrated Genome Viewer software and showing next generation sequencing reads for animals with previously unreported SNPs affecting the TMEM154 coding sequence. Numbers shown on the reads indicated the most distal identification number on the read name when viewed in the IGV software. Direct public links to these data are provided: OCAN1 F74/F74, OCAN2 F74/F74, OCAN3F74/F74, ODAL2 I74/F74, CHA02 A13/V13, BSI4 I102/T102, BSI4N70/I70, ODAL2 E31/Q31.