Differential expression of genes in three Cngb1 knockout mouse models.

Abstract

Purpose : To identify early changes in gene expression, prior to the onset of degeneration, in mouse models of retinal degeneration caused by defects in the Cngb1 locus that encodes the cGMP-gated cation channel beta-subunit and soluble proteins GARP1 and GARP2.

Methods : Genotypes used were a null allele Cngb1 X1 KO (X1 KO; 5 mo., 3 females) lacking expression of GARP1, GARP2 and CNGB1A; Cngb1 X1, Tb (X1 Tb; 6 mo., 3 males) mice that contain a transgene expressing an N-terminally truncated rod channel beta subunit; a GARP2 KO (G2 KO; 7 mo., 3 males), and WT C57Bl/6J (WT; 7.5 mo. 2 females, 1 male). RNA was isolated from retinas (n=3) using a standard procedure. Nanostring direct RNA labelling and detection was performed with 100-200 ng RNA to generate datasets from a custom gene set of 456 genes including ~100 vision related genes. nSolver 4.0.70, and nCounter Advanced 2.0.115 Analysis Software (Nanostring Technologies, Inc.) were used to analyze the data. P-values were adjusted for false discovery rates using the Benjamini-Yekutiele procedure.

Results : Twelve genes were identified that show a log₂, 1.5 fold or greater up (7 genes) or down (5 genes) regulation in X1 KO and X1 Tb RNA isolates. As expected, GARP1, GARP2, and CNGB1A controls showed greatly reduced expression (-5 to -11 fold), while XIST1 was consistently reduced in samples from males (-11 fold). Pttg1, Rec8, St6galnac2, Sugct, and Syt15 (were all up regulated 1.5 to 6.4 fold; p-value ≤0.05) in all three experimental genotypes. Cmtm5 and Impg2 were upregulated (1.5 and 1.6 fold; p-values ≤ 0.05) and Guca1b (GCAP2) and Prss56 were down regulated (1.8 and 3.1 fold; p-values ≤ 0.05) in X1 and X1 Tb samples. The genes identified function in pathways involved in phototransduction, cilium function, synaptic transmission, and chromosome dynamics.

Conclusions : Two of the genes identified are known RP genes (RP48 and RP56) that function in the phototransduction cascade (GUCA1B; GCAP2) or the interphotoreceptor matrix (IMPG2). A common theme of most identified genes is calcium regulation and participation in signal transduction pathways. The genes identified may encode interacting binding partners for the channel beta-subunit or GARPs.

This is a 2020 ARVO Annual Meeting abstract.

 

X1 vs WT volcano plot displaying each gene's -log10(p-value) and log2 fold change. Highly statistically significant genes fall at the top of the plot above the horizontal lines, and highly differentially expressed genes fall to either side.