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Iodothyronine deiodinases (Ds) mediate thyroid hormone (TH) action. They catalyze triiodothyronine (T3) production and degradation via, respectively; outer (ORD) and inner (IRD) ring deiodination. Type 1 D (D1) has a relatively high Km for substrates thyroxine (T4) and reverse T3 (rT3), catalyzes both ORD and IRD and is inhibited by propylthiouracil (PTU). Although no mutations in DIO1 gene have been reported so far in humans, based on the D1-deficient C3H mouse and the heterozygous Dio1 knockout mice, the expected phenotype of D1 loss-of-function is higher serum rT3 and slightly elevated T4. Our objective was to identify new gene defects causing unexplained and discrepant thyroid function tests using whole-exome sequencing (WES). Exons and splice sites are captured with Agilent SureSelectXT and sequenced in Illumina HiSeq2500. Data are analyzed with BWA, GATK, and ANNOVAR applications for alignment, variant calling and annotation, respectively. Sanger sequencing is used to confirm and segregate WES variants in families. A heterozygous pathogenic missense variant in the DIO1 gene (c.282C>A:p.N94K; N-linker) was identified in four family members with relatively higher serum rT3, T4, and free T4 than unaffected relatives, and normal TSH. We identified a second heterozygous pathogenic mutation in the DIO1 (c.603G>A:p.M201I; thioredoxin-fold) in a second family. Two affected individuals presented slightly elevated TSH, higher serum rT3, normal T4 levels, while the T3/T4 ratio was lower compared to unaffected members. To assess the functional activity of the mutant D1 protein, human embryonic kidney epithelial cells (HEK293) were transiently co-transfected with pcDNA3 plasmids expressing pD1-WT, pD1-N94K or pD1-M201I, and pGFP as transfection control. In assays performed with 1μM 125I-T4, the catalytic activities of pD1-N94K and pD1-M201I were 44.7% and 54.1% lower as compared to pD1-WT, respectively. To study the enzyme kinetics, the D1 assay was repeated in the presence of increasing 125I-T4 concentrations (0.1-20μM). The enzymatic activity assays revealed similar Vmax for pD1-N94K and pD1-M201I mutants compared to pD1-WT (VmaxpD1-N94K=53.7 vs VmaxpD1-WT=40.9 and VmaxpD1-M201I=58.8 vs VmaxpD1-WT=42), but higher Michaelis constant (Km) than pD1-WT (KmpD1-N94K=16.4 vs KmpD1-WT=6 and KmpD1-M201I=21.4 vs KmpD1-WT=6.9), which demonstrates a reduced affinity for T4. In conclusion, we report the first DIO1 mutations in humans with serum thyroid tests suggestive of TH metabolism defects. These mutations affect the catalytic activity of the D1, demonstrating impaired functional activity of the mutant enzymes and consequently altering TH metabolism.

OR01-01 Human Type 1 Iodothyronine Deiodinase (DIO1) Mutations Cause Abnormal Thyroid Hormone Metabolism