Daniela Choukair1, Birgit Eberle2, Philipp Vick3, Pia Hermanns4, Birgit Weiss2, Nagarajan Paramasivam5, Matthias Schlesner6, Katharina Lornsen7, Ralph Roeth2, Carina Klutmann4, Jennifer Kreis3, Georg F Hoffmann8, Joachim Pohlenz4, Gudrun A Rappold2, Markus Bettendorf8. 1. Division of Paediatric Endocrinology, Children's Hospital, University of Heidelberg, Heidelberg, Germany, daniela.choukair@med.uni-heidelberg.de. 2. Department of Human Molecular Genetics, University of Heidelberg, Heidelberg, Germany. 3. Department of Zoology, University of Hohenheim, Stuttgart, Germany. 4. Division of Paediatric Endocrinology, Children's Hospital, University of Mainz, Mainz, Germany. 5. Theoretical Bioinformatics Division, German Cancer Research Center (DKFZ), Heidelberg, Germany. 6. Bioinformatics and Omics Data Analytics (B240), German Cancer Research Center (DKFZ), Heidelberg, Germany. 7. Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany. 8. Division of Paediatric Endocrinology, Children's Hospital, University of Heidelberg, Heidelberg, Germany.
Abstract
BACKGROUND: Congenital primary hypothyroidism (CH) is the most common endocrine disorder in neonates. METHODS: To identify novel genes, we performed whole exome sequencing (WES) in 6 patients with CH due to thyroid dysgenesis (TD). The potential effects of the most relevant variants were analyzed using in silico prediction tools. The most promising candidate gene, transient receptor potential channel 4-associated protein (TRPC4AP), was sequenced in 179 further patients with TD. Expression of TRPC4AP in human thyroid was investigated using RT-PCR. Trpc4ap- functional analysis was performed in Xenopus laevis using Morpholino (MO) antisense oligomers. RESULTS: WES identified a likely damaging mutation in TRPC4AP leading to a de novo stop codon p.Q552*. Targeted sequencing of TRPC4AP demonstrated gene variants with predicted damaging potential in 5 patients resulting each in an amino acid exchange (p.P706S, p.F729L, p.S777C, and p.N229S). We demonstrated that TRPC4AP is expressed in human thyroid gland tissue. Using Xenopus laevis, we showed that the volume of the tadpole thyroid anlage was reduced by 20% in Trpc4ap MO knockdowns compared to controls and by 41% in "Clustered Regularly Interspaced Short Palindromic Repeats"/Cas9-mediated gene knockout experiments. DISCUSSION: A recognized interaction of TRPC4AP and the NF-kappa-B-essential-modulator encoded by IKBKG gene was identified by IPA analysis. IKBKG plays a role in activation of the NF-κB-signaling pathway and regulates genes involved in proliferation and survival of thyrocytes and expression of key enzymes of thyroid hormone synthesis. CONCLUSION: TRPC4AP was identified as a novel candidate gene in TD, but further studies are needed to validate its role in thyroid function.
BACKGROUND:Congenital primary hypothyroidism (CH) is the most common endocrine disorder in neonates. METHODS: To identify novel genes, we performed whole exome sequencing (WES) in 6 patients with CH due to thyroid dysgenesis (TD). The potential effects of the most relevant variants were analyzed using in silico prediction tools. The most promising candidate gene, transient receptor potential channel 4-associated protein (TRPC4AP), was sequenced in 179 further patients with TD. Expression of TRPC4AP in human thyroid was investigated using RT-PCR. Trpc4ap- functional analysis was performed in Xenopus laevis using Morpholino (MO) antisense oligomers. RESULTS: WES identified a likely damaging mutation in TRPC4AP leading to a de novo stop codon p.Q552*. Targeted sequencing of TRPC4AP demonstrated gene variants with predicted damaging potential in 5 patients resulting each in an amino acid exchange (p.P706S, p.F729L, p.S777C, and p.N229S). We demonstrated that TRPC4AP is expressed in human thyroid gland tissue. Using Xenopus laevis, we showed that the volume of the tadpole thyroid anlage was reduced by 20% in Trpc4ap MO knockdowns compared to controls and by 41% in "Clustered Regularly Interspaced Short Palindromic Repeats"/Cas9-mediated gene knockout experiments. DISCUSSION: A recognized interaction of TRPC4AP and the NF-kappa-B-essential-modulator encoded by IKBKG gene was identified by IPA analysis. IKBKG plays a role in activation of the NF-κB-signaling pathway and regulates genes involved in proliferation and survival of thyrocytes and expression of key enzymes of thyroid hormone synthesis. CONCLUSION:TRPC4AP was identified as a novel candidate gene in TD, but further studies are needed to validate its role in thyroid function.