Reduced lentivirus susceptibility in sheep with TMEM154 mutations

Abstract

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Figure 1. Seroprevalence of OPP in a research flock in 2003.

Panel A, distribution of OPP serologic status by age class: shaded bars, OPP-negative; black bars, OPP-positive. The data include 3037 females and 508 males. Panel B, relationships between average seroprevalence, average age, and breed composition. Panel C, distribution of CAEV cELISA assay values in 736 ewes 5 years and older: shaded bars, OPP-negative; black bars, OPP-positive; white bars, sheep comprising 69 matched case-control pairs of 5- to 9-year-old ewes. The percent inhibition in cELISA assay values are those reported by the diagnostic testing laboratory and included negative values.

Figure 2. GWAS and fine-mapping of TMEM154.

Panel A, relative genome position of 50,614 SNPs from the OvineSNP50 BeadChip and their unadjusted p-values for association with serologic status in 69 matched case-control pairs of 5- to 9-year-old ewes. Inset, Q-Q plot of the distribution of the test statistics. Panel B, relative genome position of 128 de novo SNPs identified in case-control sheep and their unadjusted p-values for association: circles, de novo SNPs; blue diamonds, SNPs from the OvineSNP50 BeadChip; blue bar, a 78 kb region of genomic DNA sequence containing the complete predicted TMEM154 gene; grey arrows, predicted gene regions; vertical solid bars, predicted exons; horizontal shaded bars, ovine BAC clones. Oligonucleotides for TMEM154 PCR and DNA sequencing are listed in Table S4.

Figure 3. Physical maps, amino acid sequence comparison, and median-joining network of ovine TMEM154-encoded polypeptide isoforms.

Panel A, genomic DNA and mRNA maps with missense SNPs and frameshift deletion polymorphisms: orange shading, 5′ and 3′ untranslated regions of exons; blue shading, coding regions of exons; grey shading, intron or intergenic regions. The codon polymorphism sequences are as follows: R4A^Δ, Cgc→[1 bp “c” deletion]gcg; L14H, ctt→cat; T25I, acc→atc; D33N, gac→aac; E35K, gaa→aaa; T44M, acg→atg; N70I, aat→att; E82Y^Δ, gag→[7 bp “gagttta” deletion]tat. The signal peptide was predicted with SignalP 3.0 ; the extracellular, transmembrane, and cytoplasmic domains were predicted by comparison with human TMEM154 at www.uniprot.org. Panel B, a comparison of ovine non-synonymous TMEM154 polymorphisms in exons 1 and 2 to those in related species. Polypeptide sequences for domestic sheep, bighorn sheep, domestic goat, mountain goat, muskox, cattle, gaur, yak, bison, water buffalo, caribou, elk, moose, red deer, fallow deer, mule deer, and white-tailed deer were deduced from Sanger sequencing of genomic DNA fragments amplified with ovine PCR primers. Amino acid sequences of the remaining species were obtained from Ensemble . Symbols are as follows: dot, amino acid residues identical to those in sheep haplotype 3; dash, polypeptide region not present in, or not comparable to sheep; nd, not determined. Panel C, a median-joining network of TMEM154 haplotypes encoding polypeptide isoforms . The areas of the black and white circles are proportional to the haplotype allele frequencies in 5094 sheep. The symbols are as follows: pink and blue shaded areas, full-length TMEM154 haplotypes with E and K at position 35, respectively; white circle, non-risk factor; black circles, risk factors; green circles, risk factor status unknown; shaded square, TMEM154 haplotype predicted to have occurred but unobserved in the animals tested.