Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation

Abstract

Recently, a consanguineous family was identified in Israel with three children affected by Infantile Nystagmus and Foveal Hypoplasia, following an autosomal recessive mode of inheritance. A homozygous stop mutation c.1861C > T; p.Q621^∗ in the aryl hydrocarbon receptor (AHR) gene (AHR; MIM 600253) was identified that co-segregated with the disease in the larger family. AHR is the first gene to be identified causing an autosomal recessive Infantile Nystagmus-related disease in humans. The goal of this study is to delineate the molecular basis of this newly discovered human genetic disorder associated with a rare AHR gene mutation. The gene and protein expression levels of AHR and selected AHR targets from leukocyte cultures of healthy subjects and the patients were analyzed. We observed significant variation between mRNA and protein expression of CYP1A1, CYP1B1, and TiPARP under rest and AHR-induced conditions. The CYP1A1 enzymatic activity in induced leukocytes also differs significantly between the patients and healthy volunteers. Intriguingly, the heterozygous subjects demonstrate CYP1A1 and TiPARP gene and protein expression similar to homozygous patients. In contrast, CYP1B1 inducibility and expression vary between hetero- and homozygous subjects. Similarity and differences in gene and protein expression between heterozygotes and homozygous patients can give us a hint as to which metabolic pathway/s might be involved in the Nystagmus etiology. Thus, we have a unique human model for AHR deficiency that will allow us the opportunity to study the biochemical basis of this rare human mutation, as well as the involvement of AHR in other physiological processes.

Keywords: CYP1A1; CYP1B1; TiPARP; aryl hydrocarbon receptor; gene expression; human mutation; infantile nystagmus; protein expression.

FIGURE 1

AHR mRNA (A) and protein (B,C) expression. In all graphs, the columns correspond to lysates of non-induced leukocyte cultures (white columns) or lysates of BA-induced cultures (BA, black columns). (A) Relative AHR mRNA expression was examined by qRT PCR using GAPDH as an internal control. (B) AHR protein expression was detected by western blotting and detection with an antibody against a C-terminal epitope. (C) AHR protein expression was detected by western blotting and detection with an antibody against an N-terminal epitope. In all experiments, leukocyte culture lysates were run on 10% SDS PAGE, transferred to nitrocellulose and probed with specific AHR C-terminal (B) or N-terminal (C) antibodies. Band densities were normalized to β-Actin and quantified as a fraction of non-treated WT samples. Data were obtained separately from either patients or healthy subjects and were averaged. Data are shown as mean ± SD, n = 4 technical replicates for each of three healthy volunteers (Heal), two heterozygous patients (Het), and three homozygous patients (Hom). Insets show a representative AHR western blot obtained using a. LI-COR Odyssey IR scanner. Upper bands correspond to AHR and lower bands correspond to the β-actin loading control. A two-tailed t-test analysis of statistical significance was done; p < 0.01* and p < 0.05** refer to variances between homozygous patients vs. healthy control samples.

FIGURE 2

mRNA and protein expression of AHR target genes. CYP1A1 (A,B), CYP1B1 (C,D), and TiPARP (E,F). In all graphs, the columns correspond to lysates of non-induced leukocyte cultures (white columns) or lysates of BA-induced cultures (BA, black columns). (A) Relative CYP1A1 mRNA expression was examined by qRT PCR using GAPDH as an internal control. (B) Relative CYP1A1 protein expression was determined by western blotting with an antibody against CYP1A1. (C) Relative CYP1B1 mRNA expression was examined by qRT PCR with GAPDH as internal control. (D) CYP1B1 protein expression was determined by western blotting with CYP1B1 antibodies. (E) Relative TiPARP mRNA expression was examined by qRT PCR using GAPDH as internal control. (F) TiPARP protein expression was detected with PARP antibodies. The leukocyte culture lysates were run on 10% SDS PAGE, transferred to nitrocellulose and probed with specific antibodies as described in the “Materials and Methods” section. Band densities were normalized to the β-Actin loading control and quantified as a fraction of non-treated WT. Data were obtained separately from either patients or healthy subjects and were averaged. Data are shown as mean ± SD, n = 4 technical replicates for each of three healthy volunteers, two heterozygous patients (Het), and three homozygous patients (Hom). Insets show a representative western blot obtained using a LI-COR Odyssey IR scanner. Upper bands correspond to tested protein and lower bands correspond to β-Actin loading control. A two-tailed t-test analysis of statistical significance was done; p < 0.01* and p < 0.05** refer variances between homozygous patients vs. healthy control samples.

FIGURE 3

mRNA and Protein expression of AHR target genes. ARNT (A,B) and AHR-R (C,D). In all graphs, the columns correspond to lysates of non-induced leukocyte cultures (white columns) or lysates of BA-induced cultures (BA, black columns). (A) Relative ARNT mRNA expression was examined by qRT PCR using GAPDH as an internal control. (B) ARNT protein expression was detected by western blotting and detection with ARNT antibodies. (C) Relative AHR-R mRNA expression was examined by qRT PCR using GAPDH as an internal control. (D) AHR-R protein expression was detected by western blotting and detection with AHR-R antibodies. The leukocyte culture lysates were run on 10% SDS PAGE, transferred to nitrocellulose and probed with specific antibodies as described in the “Materials and Methods” section. Bands were normalized to the β-Actin loading control and quantified as a fraction of non-treated WT. Data were obtained separately for either patient or healthy subject and were averaged. Data are shown as mean ± SD, n = 4 technical replicates for each of three healthy volunteers, two heterozygous subjects (Het), and three homozygous patients (Hom). Insets show a representative western blot obtained using a LI-COR Odyssey IR scanner. Upper bands correspond to tested protein and lower bands correspond to β-Actin loading control. A two-tailed t-test analysis of statistical significance was done; p < 0.01* and p < 0.05** refer variances between homozygous patients vs. healthy control samples.