SAT-286 TSH Synthesis and Secretion Are Unperturbed in Male IRS4 Knockout Mice

Abstract

It was recently reported that mutations in the insulin receptor substrate 4 (IRS4) gene cause a novel form of X-linked congenital central hypothyroidism (OMIM 300904). To date, four different mutations, three frameshift and one nonsense, have been reported, with two affected male patients showing decreased basal, pulsatile, and total thyroid-stimulation hormone (TSH) secretion (PMID 30061370).Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and their downstream effectors. IRS4/Irs4 expression is enriched in the pituitary; however, its role in the hypothalamic-pituitary-thyroid (HPT) axis has not been studied in detail.We generated novel whole-body Irs4-knockout mouse lines using CRISPR-Cas9. A specific guide RNA was used to target the Cas9 enzyme to the 5’ end of the single exon Irs4 gene. A two-nucleotide deletion was introduced into Irs4, resulting in a frameshift and premature stop codon. We hypothesized that like IRS4 deficient patients, these mice would exhibit central hypothyroidism. Given that Irs4 is X-linked, we focused our initial characterization on males.Under normal laboratory conditions, Irs4 knockout mice do not exhibit differences in pituitary expression of Tshb, which encodes one of the subunits of the TSH heterodimer. Expression of the gene encoding the thyrotropin-releasing hormone (TRH) receptor, Trhr1, is also unperturbed in these knockout mice. Additionally, there are no differences in their serum thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine). When Irs4 knockout males were placed on a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks and rendered hypothyroid, their serum TSH increased similarly to wild-type males. Overall, Irs4 knockout males do not exhibit central hypothyroidism or phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these mice.