Homozygous loss-of-function mutations in SLC26A7 cause goitrous congenital hypothyroidism

Abstract

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.

Keywords: Endocrinology; Genetics; Molecular genetics; Monogenic diseases; Thyroid disease.

Figure 1. Thyroid hormone biosynthesis pathway.

Schematic depicting the thyroid follicular cell and key components of the thyroid hormone biosynthesis pathway. The major steps in iodide metabolism (uptake, efflux, organification, and recycling) are shown. In murine thyroid, Slc26a7 localizes intracellularly and to the basal membrane; given its known function as a chloride-bicarbonate transporter in other cell types, Slc26a7 may alter intracellular pH, thereby altering iodide uptake or iodide organification as we have observed in murine or human contexts, respectively. DIT, diiodothyronine; MIT, monoiodothyronine.

Figure 2. Clinical genotype and phenotype data for kindreds harboring SLC26A7 mutations, and functional characterization of wild-type and mutant SLC26A7.

(A) Pedigrees of 6 kindreds illustrating cases with congenital hypothyroidism (CH) and unaffected relatives for whom genetic data were available. Thyroid hormone measurements refer to venous measurements made at diagnosis of CH or following genetic evaluation. Reference ranges: AII.i, iii, iv, v thyroid-stimulating hormone (TSH) 0.9–3.5 mU/l, free thyroxine (FT4) 1.1–1.8 ng/dl; AII.ii TSH 0.6–4.8 mU/l, FT4 0.76–1.5 ng/dl; AII.vi. vii, viii, ix TSH 0.4–3.5 mU/l, FT4 0.83–1.69 ng/dl; BII.i TSH 0.7–5.3 mU/l, FT4 1.1–1.8 ng/dl; CII.i TSH 0.6–4.8 mU/l, FT4 0.8–1.5 ng/dl; CII.ii TSH 0.4–3.5 mU/l, FT4 0.8–1.9 ng/dl; DII.i, ii, EII.i, ii, FII.i, ii TSH 0.35–40 mU/l, FT4 0.7–1.5 ng/dl. Double horizontal lines indicate consanguinity. FT4: multiply by 12.87 to convert to pmol/l. (B) Quantitative RT-PCR analysis of SLC26A7 mRNA expression in a human tissue library relative to cyclophilin; horizontal bars denote mean and vertical bars standard error of the mean. (C) Homology modeling predicting the protein consequences of the SLC26A7 p.F631Lfs*8 mutation. The deleted protein region is shown in red. (D) HEK293 cells transfected with GFP-tagged wild-type and F631Lfs*8 SLC26A7 demonstrate localization of wild-type protein to the plasma membrane, whereas the mutant remains intracellular (total original magnification, ×63). (E) Box-and-whisker plots for time-dependent iodide efflux from HEK293 cells cotransfected with NIS and pCDNA (white), pendrin (gray), or SLC26A7 (stippled black); n = 5 experiments. Boxes extend from the 25th to 75th percentiles, the horizontal line represents the median, and the vertical bars represent the minimum and maximum values.

Figure 3. Characterization of the thyroid phenotype in Slc26a7-null mice and evaluation of murine thyroidal Slc26a7 expression.

(A) Box-and-whisker plots for log₁₀TSH, T4, and T3 measurements in male wild-type (WT, white), and Slc26a7-null mice (KO, stippled black) at 14, 60, and 90 days of age; n = 6–12 mice of each genotype. Boxes extend from the 25th to 75th percentiles, the horizontal line represents the median, and the vertical bars represent the minimum and maximum values. P values were calculated using an unpaired 2-tailed Student’s t test. ***P < 0.0005. (B) Quantitative RT-PCR analysis of Slc26a7 mRNA expression in a murine tissue library relative to cyclophilin. Horizontal bars represent the mean and error bars represent the standard error of the mean. (C) Photographic representation of the thyroid gland in a male wild-type and Slc26a7-null mouse age 90 days, demonstrating goiter in the Slc26a7-null mouse. Box-and-whisker plots below quantify mean thyroid weight/body weight in 5 WT (white), 18 heterozygous (Het, gray), and 8 Slc26a7-null male mice (KO, stippled black) at P14. P values were calculated using 1-way ANOVA with the Newman-Keuls multiple-comparisons test. **P < 0.005, ***P < 0.0005. (D) Hematoxylin and eosin–stained thyroid sections from a male Slc26a7-null (KO) mouse compared with a heterozygous littermate (Het) as control age 60 days. Images are representative of sections reviewed from 3 separate goitrous mice and demonstrate enlarged, colloid-filled follicles with normal-tall follicular cells. Scale bars: 500 μm (top left) and 100 μm. (E) Representative thyroid sections from 2 different Slc26a7-FLAG male mice and a wild-type mouse age 75 days, stained with anti-FLAG antibody or preimmune serum (control). Arrows denote intracellular and basolateral localization of SLC26A7. Scale bars: 10 μm. NS, not significant.

Figure 4. Intrathyroidal hormone measurements in male Slc26a7-null mice.

Box-and-whisker plots for intrathyroidal TG-bound and free (F) T4 and T3 levels in male wild-type (WT, white) and homozygous Slc26a7-null mice (KO, stippled black) age 90 days. Boxes extend from the 25th to 75th percentiles, the horizontal line represents the median, and the vertical bars represent the minimum and maximum values. Measurements were made from 6–10 mice of each genotype. P values were calculated using an unpaired 2-tailed Student’s t test. ***P < 0.0005.