Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse

Abstract

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ(2)=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

Figure 1. Congenital stationary night blindness is associated with homozygosity for leopard complex spotting in horses.

(A) lp/lp horses do not display a leopard complex spotting pattern and are not night blind as evidenced by normal ERGs. (B) LP/lp horses display one of several characteristic spotting patterns that vary in the amount of un-pigmented hairs in the coat and have pigmented spots (“leopard spots”) in the un-pigmented area. These horses are not night blind as evidenced by normal ERGs. (C) LP/LP horses displaying the characteristic homozygous patterns with varying amounts of white on the coat with few to no “leopard spots”. These horses all have CSNB as evidenced by the “negative” ERG in which the b wave is absent.

Figure 2. LP-associated characteristics.

In addition to coat color spotting patterns LP also causes four other associated pigmentation characteristics, readily visible white sclera (as shown in A) striped hooves, which are bands of pigment on the hoof alternating with unpigmented bands (as shown in B), mottling, which is pink skin with spots of pigment that occurs around the anus, genitalia (C), muzzle (D), and eyes (E), and varnish roaning, the progressive loss of pigment throughout the coat while retaining pigment on boney surfaces; as shown here on the face, hips, and lower legs (E).

Figure 3. RNA sequencing reads mapped to TRPM1.

RNA-Seq reads from LP/LP CSNB affected and lp/lp CSNB unaffected retina RNA, as well as, from LP/LP, LP/lp and lp/lp skin RNA. ECA1 Mb, TRPM1 exon position, location of the insertion, and the number of reads for each sample are presented. (A) exon 0-27 illustrating that in LP/LP samples transcripts were not detected in exons 3’ of the insertion in intron 1. (B) exon 0, 1, 1’, 2 and 3 of TRPM1 illustrating that transcripts from exon 0 (yellow highlight) were detected in retinal samples but not skin while transcripts from exon 1’ (green highlight) were not detected in any of the horse samples, and finally transcripts were detected that mapped to intron 1 of LP/LP and LP/lp samples allowing for the identification of the insertion (denoted by red line).

Figure 4. LTR insertion in TRPM1 causes premature poly-adenylation.

(A) The 1378bp LTR insertion (ECA1g. 108,297,929_108,297,930 ins1378) is flanked by 6 bp regions of micro-homology which are highlighted in yellow. Poly-adenylation signal sequences are highlighted in green. The CA dinucleotide cleavage and poly-adenylation site is highlighted in red. (B) poly(A) tail detected by 3’RACE in LP/LP skin and (C) CSNB LP/LP retina samples. Sequence represented is from poly-adenylation signal sequence AGUAAA through poly(A) tail.

Figure 5. Skin biopsies and melanocyte cultures for LP/LP and lp/lp samples.

Coat color phenotypes of (A) LP/LP horse and (B) lp/lp . Close-up views of the biopsied skin from LP/LP horse (C) unpigmented samples and (E) pigmented spot. Close up view of (D) pigmented area and (F) biopsied skin from lp/lp horse taken from approximately the same location as the LP/LP pigmented spot on the rump. Morphology of (G) LP/LP and (H) lp/lp melanocytes in culture at passage 7 and 6 respectively. Culture images were captured on Nikon microscope with 20x objective.

Figure 6. LP/LP and lp/lp equine melanocyte culture data.

(A) Ultrastructure of melanocytes from human and horse epidermis at passages 3, 5, and 2 (humans, lp/lp and LP/LP respectively) were plated on glass coverslips and processed for transmission electron microscopy. Insets show melanosome morphology at higher magnification. Scale bar: 2μm. (B) qRT-PCR analyses of TRPM1, mRNA expression in LP/LP and wild type lp/lp melanocytes. TRPM1 was differentially expressed between LP/LP and lp/lp melanocytes (*p=0.01). (C) Fluorescence ratio change in lp/lp and pigmented LP/LP melanocytes. LP/LP cells had significantly lower peak Ca²⁺ uptake (***p<0.001).

Figure 7. The role of TRPM1 in night vision.

Night vision is controlled by ON visual pathway which comprises both retinal rod and ON-bipolar cells and involves the neurotransmitter glutamate, its receptor mGluR6, the G protein Gα_o, TRPM1, and Ca²⁺. Photon absorption by the rod cell causes the rod cell to hyperpolarize (resulting in the “a” wave of the ERG) and decreases the concentration of glutamate being released by that cell. This leads to the opening of the calcium ion channel, TRPM1, and results in the depolarization of the ON bipolar cell (“b” wave on the ERG). In the absence of photon activation, low light conditions, the rod cell is depolarized leading to the release of glutamate and the closing of TRPM1. This results in the hyperpolarization of the ON-bipolar cell. In contrast, in LP/LP CSNB horses TRPM1 is unavailable to respond to changes in glutamate concentration and thus the ON-bipolar cells remain hyperpolarized in response to light. The b-wave is absent, so the ERG appears “negative” as it is only the a-wave (the rod cell response) that is detected.