The patient was a 19-year-old Tunisian man who moved to Italy in 2021. Two months after his arrival, he experienced symptomatic neck enlargement, which was confirmed as a diffuse thyroid goiter by ultrasound (US). Initial blood tests revealed severe primary hypothyroidism (FT4 0.06 ng/dL n.v. 0.90–1.70, FT3 0.50 pg/mL n.v 2.10–4.30, TSH 396.80 µIU/mL n.v. 0.27–4.20) and the patient was promptly referred to our Center for further investigations. Notably, he exhibited no signs of mental retardation or explicit hypothyroidism symptoms. He reported no personal or familial history of thyroid disease or medication use. At the time of admission, his height was 1,76 m, his weight 62,5 kg, and his body mass index 20,2 kg/m2, indicative of normal harmonious growth. Physical examination showed a myxedematous face, with periorbital and labial edema, and neck examination identified a large diffuse goiter. No other signs of hypothyroidism, such as macroglossia, umbilical hernia or cutaneous annexes abnormalities were detected. The patient presented with severe primary hypothyroidism, as indicated by thyroid function tests (Table 1), and high levels of transaminases and lactate dehydrogenase, compatible with the state of hypothyroidism (data not shown). Ultrasound confirmed a diffuse goiter (thyroid volume 94 ml). A tru-cut biopsy showed a micro-macrofollicular thyroid with hyperplastic features. Autoimmune and infiltrative thyroid diseases were excluded, and no iatrogenic/toxic implication was detected. Thyroid scintigraphy with 131I showed increased uptake (81.6% and 98.3% after 3 and 24 h, respectively) (Fig. 1a) and the perchlorate-induced iodide discharge test indicated a 31.8% discharge rate (Fig. 1b), suggesting a partial organification defect. L-thyroxine (LT4) replacement therapy was initiated and titrated, resulting in euthyroidism and goiter reduction (thyroid volume 41 ml). Urinary parameters were also evaluated to assess potential kidney-related fluids and electrolytes alterations, but no significant abnormalities were observed (Table 2). The patient’s brother, the only relative available for examination, was clinically and biochemically euthyroid (FT4 1.06 ng/dL; FT3 5.16 pg/mL; TSH 1.1 mIU/mL; AbTPO and AbTg undetectable).

Thyroid scintigraphy with 131I (a) and perchlorate discharge test (b)

Thyroid morphology and function were evaluated using thyroid ultrasound, perchlorate discharge test as well as measurements of serum FT4, FT3, TSH, anti-TG and anti-TPO antibodies, as described [11]. A twenty-four-hour urine collection was performed to determine urine ionic composition via potentiometric system. Written informed consent for genetic analyses and for the scientific use of data were obtained from the proband and his brother in accordance with the Declaration of Helsinki and subsequent amendments (Good Clinical Practice guidelines).

Targeted NGS was conducted to investigate the cause of the goitrous hypothyroidism in our patient. We designed a custom panel targeting 34 genes involved in primary CH pathogenesis [10, 12,13,14,15,16] (Table 3). Genomic DNA was isolated from peripheral blood cells. For library preparation, we used the SureSelect QXT Reagent Kit (Agilent Technologies Inc., Santa Clara, CA, USA). Custom capture probes were then hybridized to the target sequences of the library for sequence enrichment. The enriched library was amplified using dual indexing primers. Equimolar amounts of multiple libraries were pooled into a single sample and sequenced on an Illumina MiSeq Dx System platform (Illumina Inc, San Diego, CA, USA) using the MiSeq Reagent Nano Kit V2 300 Cycles (2 × 151 bp paired-end run). The MiSeq platform generated a pair of FastQ files per sample suitable for secondary analysis with SureCall NGS software version 4.2.1 (Agilent Technologies Inc., Santa Clara, CA, USA). We performed in silico analysis of clinically relevant variants using the free web-based softwares Mutation Taster, PROVEAN and MutPred to estimate the variant's impact on the gene product. The selected variants were validated by Sanger sequencing [17]. Segregation analysis was subsequently performed.

SureCall software identified 6 pathogenic gene variants, five of which were UTR or non-coding transcript variants for the IYD and HOXB3 genes. The sixth variant, identified as chr8:92406214 AC > A, was located in the coding region of the SLC26A7 gene. This variant, registered as dbSNP ID rs768718640, affected transcripts NM_001282356, NM_001282357, NM_052832, and NM_134266, leading to a frameshift mutation with a high impact. Specifically, the homozygous deletion of the C at position 1883 of the exon 18 of the SLC26A7 gene caused a proline to glutamine substitution at position 628 of the protein, a frameshift, and a formation of a stop codon (TAA) after 11 amino acids. GnomAD Genome indicated a minor allele frequency of 0.00002833 suggesting that the variant is exceptionally rare in the general population. The new variant called c.1883delC or p.P628Qfs*11 was not present in ClinVar and was predicted to be pathogenic by MutationTaster, PROVEAN and MutPred. The Exome Aggregation Consortium accessed in January 2023 did not report homozygous cases (ExAC, https://gnomad.broadinstitute.org/) for this truncating variant. Sanger sequencing confirmed the presence of the homozygous and heterozygous P628Qfs*11 mutation in the proband and the brother, respectively. The proband family tree and sequence electropherograms were illustrated in Fig. 2.

Sanger sequencing and segregation analysis of the pathogenic variant of the SLC26A7 gene. Patient’s sequence electropherogram showing the homozygous deletion of the C (a), brother’s sequence electropherogram showing the heterozygous deletion of the C (b) and family pedigree (c). Empty symbols with a “?” inside represent family members not investigated

Given that the 30 μg iodide/day supplementation partially reversed hypothyroidism in Slc26a7-null male mice [8], we suggested to the patient a treatment trial with 1 mg iodide/day after LT4 suspension (TSH 5,72 mIU/L; FT3 5,25 ng/L; FT4 1,02 ng/dL). However, after 13 days, we ceased this treatment and reintroduced LT4 due to the decline of thyroid hormonal profile and the onset of an acute myopericarditis (TSH 17,4 mIU/L; FT4 0,65 ng/dL).