Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation

Abstract

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN (acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE. In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygous frameshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lep^ob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

Fig. 1. Schematic representation (not to scale) of the exon-intron structure of the CTBP2 genomic locus and splicing pattern.

The green boxes represent exons exclusively producing CTBP2-L. The orange boxes are exons specific for CTBP2-S, while the blue boxes indicate RIBEYE’s specific A-domain [17].

Fig. 2. Workflow of the study.

Based on previous analyses [ref for Hinney 2017, MolPsych], Ctbp2 and its respective isoforms (CTBP2-L/S and RIBEYE) were analysed in in vitro, in vivo and in silico analyses. AN anorexia nervosa, BMI body mass index, Ctbp2 C-terminal binding protein 2, GT genotyping, MS mutation screen.

Fig. 3. Gene expression profiling of Ribeye mRNA in murine hypothalamus.

A Hypothalamic expression in mice fed ad libitum (ad lib), in mice fasted for 12 h, 24 h or 36 h or in mice fasted for 36 h with subsequent re-feeding for 6 h with either a fat-free diet (FFD) or a high fat diet (HFD). B Hypothalamic expression profiles for diet-induced obesity (DIO) and age-matched lean control mice. All results were normalized to the housekeeping gene HPRT. Average of Ct values are displayed in the respective bar graphs. One-way analysis of variance (ANOVA) with Tukey’s multiple post-hoc comparison test. HPRT hypoxanthine guanine phosphoribosyltransferase 1. n number of samples.

Fig. 4. Effect of leptin administration and pair feeding of Lep^ob/ob mice on hypothalamic and midbrain Ribeye and Ctbp2 mRNA expression.

Ribeye (A) and Ctbp2 (B; all isoforms) expression analyses were performed in vehicle-treated Lep^ob/ob mice fed ad libitum (ad lib), in vehicle-treated pair-fed (PF) Lep^ob/ob mice and in leptin-treated Lep^ob/ob mice. *p < 0.05, based on a one-way ANOVA with Tukey’s multiple post-hoc comparison test. Average of Ct values are displayed in the respective bar graphs, HPRT hypoxanthine guanine phosphoribosyltransferase. n number of mice used.