Concomitant Mutations in the Thyroglobulin and SLC26A4 Genes Leading to Fetal Goiter and Congenital Hypothyroidism in a Patient With Pendred Syndrome

Abstract

We described new forms of thyroglobulin gene (TG) mutation resulting in fetal goiter and congenital hypothyroidism in a pendred syndrome (PS) patient. Fetal hypothyroidism was diagnosed, based on ultrasonographic evidence of goiter alongside with fetal blood measurement of TSH (>100 mIU/L); levothyroxine intrauterine treatment was performed. At birth, ultrasound thyroid enlargement and primary hypothyroidism were confirmed. On Day 11, hearing screening using a combination of otoacoustic emissions and auditory brainstem response revealed profound bilateral sensorineural deafness. Genetic analysis revealed TG variants [c.727C>T,p.(Àrg243Trp) and c.67l8G>T,p.(Glu2240Stop) in heterozygosis. Mutation c.164+1delG,p.(Ser55Ilefs⁣^∗11) in homozygosis of the SLC26A4 gene was also detected. The extension of the analysis to the patient's parents revealed the presence of the heterozygous variant in the SLC26A4 gene [c.164+1delG] and in the TG gene [c.727C>T] in the father, and the heterozygous variant in the SLC26A4 gene [c.164+1delG] and in the TG gene [c.6718G>T] in the mother. This is the first report associating mutations in TG with a PS patient. The combination of genetic factors likely contributed to the patient's clinical conditions, which included fetal goiter with CH and profound bilateral hearing loss, representing a rare instance of mutations linked to inherited thyroid disorders.

1. Introduction

A fetal goiter is an abnormal enlargement of the thyroid gland caused by impaired fetal thyroid hormone synthesis [1].

Congenital hypothyroidism (CH) is the most common endocrine disorder in infants, occurring in one out of 3000–4000 births. CH associated with thyroid enlargement occurs in about one out of 40,000 births, representing 10%–15% of all cases [2–4]. Early CH detection and treatment are crucial, as they can prevent serious neurodevelopmental consequences, including intellectual disability. Thyroid dyshormonogenesis (TDH) accounts for 10%–15% of all CH cases [5]. TDH results from defects at various stages of thyroid hormone synthesis. Mutations in the thyroglobulin gene (TG) are a common cause and are often associated with goiter, which may appear during fetal or neonatal life [5–9].

Pendred syndrome (PS) is an autosomal recessive disorder caused by mutations of the SLC26A4 gene and characterized by a combination of sensorineural deafness and goiter, with or without hypothyroidism [10, 11]. In addition to hearing loss and thyroid issues, people with PS may experience balance disturbances due to vestibular abnormalities. Hearing loss typically represents the earliest clinical manifestation, whereas thyroid enlargement or other thyroid abnormalities usually develop later, after the onset of deafness [10, 11].

In this report we described concomitant mutations in the TG and SLC26A4 genes leading to fetal goiter and CH in a patient with PS. An association between TG and SLC26A4 genes has not been previously described.

2. Case Presentation

A 25-years-old quintigravida of Egyptian origin, affected by insulin-dependent diabetes and subclinical nonimmune hypothyroidism, and poor obstetrical history without children, due to cervical incontinence was referred to our Fetal Therapy Unit at 20.4 weeks gestation for a fetal goiter measuring 28 mm diameter (Figure 1).

Figure 1.

Ultrasound images of fetal goiter at 20.4 weeks gestation. Panel A,B: ultrasound 2D sagittal (A) and longitudinal (B) planes; Panel (C) 3D ultrasound.

Thyroid-stimulating hormone (TSH) level on fetal blood sampling (FBS) was >100 mU/L (normal range 0.35–4.94 mU/L), while free thyroxine (fT4) was 5.1 pmol/L (normal range 9–19 pmol/L) so intrauterine treatment was initiated. Intra-amniotic injection of 100 mcg of levothyroxine once a week was performed from 22 to 33 weeks, with an increased dosage of 200 mcg of levothyroxine after the first two administrations. Serial ultrasound assessments were performed to monitor fetal growth, size of the goiter, fetal signs of cardiac dysfunctions, and the occurrence of polyhydramnios. Concurrently, the efficacy of intra-amniotic therapy was assessed by FBS and amniotic fluid TSH levels trend (amniotic fluid TSH levels at 27 weeks: 2.9 mU/L; at 29 weeks: 1.2 mU/L; at 33 weeks: 1.8 mU/L) [12]. Her Egyptian consanguineous partner has no significant past medical history.

At 38 weeks, a live female infant was delivered in good condition, with Apgar scores of 8 and 9 at 1 and 5 minutes, respectively. Birth weight of the newborn was 3020 g (p.le 54°), height was 48 cm (p.le 42°) and head circumferences was 32.9 cm (34° p.le). Initial examination was notable for a large diffuse neck swelling and general moderate hypotonia, with no other abnormal examination findings. The infant was transferred to the neonatal unit for further investigation and management. An ultrasound confirmed enlargement of both lobes of the thyroid gland, including the isthmus; the gland presents normal texture and was hyperemic.

Thyroid function tests within the first 24 h of life indicated primary hypothyroidism with TSH > 100 mIU/L (normal range 0.73–4.77 mIU/L), free thyroxine (fT4) at 6.7 pmol/L (normal range 10.5–18.8 pmol/L), and preserved free triiodothyronine (fT3) levels at 6.2 pmol/L (normal range 3.6–7.5 pmol/L), with no detectable thyroid antibodies.

Levothyroxine treatment (10 mcg/kg) was started on Day 1 of life, with progressive normalization of thyroid function tests.

Due to the association of fetal goiter with laboratory evidence of hypothyroidism, genetic studies were undertaken with written informed parental consent.

On Day 11, hearing screening using a combination of otoacoustic emissions and auditory brainstem response revealed profound bilateral sensorineural deafness. Magnetic resonance imaging showed an enlargement of the endolymphatic sac and duct, along with a large vestibular aqueduct. A cochlear implantation was planned.

The results of the TDH gene panel assessment (Next generation sequency: Custom Panel Enrichment and Nextera Flex Enrichment-Illumina) revealed new forms of heterozygous variants in the TG gene [c.727C>T,p.(Àrg243Trp) at exon 6 and c.67l8G>T, p.(Glu2240Stop) at exon 38]. A mutation c.164+1delG,p.(Ser55Ilefs⁣^∗11) in homozygosis at the exon–intron 2 junction of the SLC26A4 gene was also detected.

The SLC26A4 c.164+1delG variant is a deletion at the donor splice site of intron 2, resulting in a frameshift and premature stop codon (p.Ser55Ilefs⁣^∗11). This alteration affects the N-terminal cytoplasmic region, upstream of the first transmembrane segment, and is predicted to cause complete loss of the anion transporter domain responsible for iodide/chloride exchange. The expected outcome is a nonfunctional pendrin protein, consistent with the patient's sensorineural deafness and PS phenotype.

The TG c.727C>T (p.Arg243Trp) variant, located in exon 6, lies within the type-1 TG repeat domain, a highly conserved structural motif essential for protein folding within the endoplasmic reticulum. The substitution of a positively charged arginine with a bulky hydrophobic tryptophan likely disrupts local conformation and compromises proper folding and secretion of TG.

The TG c.6718G>T (p.Glu2240Stop) variant, in exon 38, introduces a premature termination codon within the acetylcholinesterase-like (ChEL) domain, which plays a crucial role in intracellular trafficking and secretion of TG. The resulting truncated protein lacks the C-terminal region necessary for export from the endoplasmic reticulum to the follicular lumen, thereby impairing thyroid hormone biosynthesis.

The extension of the analysis to the patient's parents revealed the presence of the heterozygous variant in the SLC26A4 gene [c.164+1delG] and in the TG gene [c.727C>T] in the father, and the heterozygous variant in the SLC26A4 gene [c.164+1delG] and in the TG gene [c.6718G>T] in the mother. The trans configuration of the TG gene mutations may be pathogenic for the patient's thyroid phenotype. The documented mutation in the SLC26A4 gene allowed the hearing loss to be classified under the diagnosis of PS. Table 1 summarizes the segregation pattern.

Table 1.

Segregation of the identified variants within the family.

Gene	Variant (cDNA/protein)	Father	Mother	Patient	Zygosity/interpretation
TG	c.727C>T/p.Arg243Trp	Heterozygous	Wild-type	Heterozygous	Paternal variant (TG#1)
TG	c.6718G>T/p.Glu2240Stop	Wild-type	Heterozygous	Heterozygous	Maternal variant (TG#2)
SLC26A4	c.164+1delG/p.Ser55Ilefs⁣∗11	Heterozygous	Heterozygous	Homozygous	Variant inherited from both parents

At the diagnosis, variant coordinates were reported according to the GRCh37/hg19 human genome assembly. To better characterize the identified variants, we also reanalyzed using the GRCh38/hg38 genome assembly and verified their presence in the main population and clinical databases (gnomAD v4.1, ClinVar, ClinGen Allele Registry, and dbSNP). The results, including allele frequencies, are summarized in Table 2.

Table 2.

Variant annotation was performed using the GRCh38/hg38 genome assembly and verified across major reference databases: gnomAD v4.1, ClinVar, ClinGen Allele Registry, and dbSNP.

Gene	Nucleotide change (cDNA)	Protein change	Genomic position (hg38)	dbSNP/ClinVar/ClinGen ID	gnomAD v4.1 (allele count/total)	Pathogenicity interpretation
TG	c.727C>T	p.Arg243Trp	chr8:132,881,951 C>T	rs565270285/ClinVar 3373484/CA4882995	21/1,613,456 (AF ≈ 1.3 × 10−5)	Likely pathogenic (missense, rare, conserved residue)
TG	c.6718G>T	p.Glu2240Ter (stop)	chr8:132,868,206 G>T	Not reported	Not found	Pathogenic (null variant in critical domain)
SLC26A4	c.164+1delG	p.Ser55Ilefs⁣∗11 (splice donor)	chr7:107,661,805 delG	ClinVar VCV000188838	Not found	Pathogenic (established cause of Pendred syndrome)

Note: If a variant was not indexed in these databases, this is explicitly indicated. Pathogenicity classification was assigned according to ACMG/AMP 2015 guidelines, categorizing variants as pathogenic, likely pathogenic, variant of uncertain significance (VUS), or benign. For each variant, the following information is provided: genomic coordinates, database identifiers, allele frequency (AF) when available.

3. Discussion

We described a case involving concomitant mutations in the TG and SLC26A4 genes, resulting in fetal goiter and CH in a patient with PS. Although these genes are usually associated with distinct disorders, their co-occurrence suggests a synergistic effect at the molecular level. The SLC26A4 mutation disrupts the anion transport domain, while the TG variants affect folding and secretion domains, both essential for proper protein function. These combined alterations likely underlie the patient's clinical presentation.

Fetal goiter occurs in ~1 in 5000 births and may serve as a prenatal marker of CH resulting from TDH. It can lead to significant perinatal complications, such as polyhydramnios, intrauterine death, preterm birth, labor dystocia, and neonatal asphyxia. Intra-amniotic levothyroxine administration has been suggested as a therapeutic option to mitigate these risks [12].

It should be noticed that there is no consensus regarding who should receive treatment or when the treatment should be started, which hormone to use, appropriate dose, the number of administrations, and the interval between them [13, 14]. The primary aim of prenatal treatment consists in the reduction of perinatal complications enabling pregnancy to come to term [14]. The literature has demonstrated contrasting efficacy in reducing goiter size [1, 13, 14]. In our case, we did not achieve an absolute fetal goiter size reduction, although the relative proportion of goiter significantly reduced as fetal growth occurred and the neonate did not present distress respiratory at birth due to airway compression.

Despite in utero therapy, our case had a severe hypothyroidism at birth, similarly to other previously published cases [1]. The timing of the latest injection before birth has been reported to be a key factor influencing newborn thyroid status [14]. In our case, the decision not to repeat the intra-amniotic injection in the late third trimester was based on the reassuring amniotic fluid TSH levels trend and took into account the benefit-to-risk analysis of these repeated procedures, which are associated with increased risk of preterm labor and chorioamnionitis, especially amplified in our patient due to her previous obstetrical history.

To date, mutations in TG, thyroid peroxidase (TPO), DUOX maturation factor 2 (DUOXA2), and sodium iodide symporter (NIS) have been identified in few dyshormonogenetic fetal goiter patients [1, 14–27].

Specifically, in addition to TPO gene mutations [9, 17, 20], goiter is a common manifestation TG [21–27] gene mutations.

TG gene occur with an estimated frequency of at least 1 in 100,000 and are generally inherited in an autosomal recessive manner. Affected individuals may present a wide spectrum of thyroid dysfunction, ranging from severe CH to euthyroid goiter, depending on the specific mutation and its impact on TG synthesis and secretion [5]. Goiter often appears in the neonatal period, though it can develop later; in a small minority of cases, fetal goiter is observed. In our case, absence of earlier ultrasound data prevents definitive conclusions about the onset of goitrogenesis. However, the goiter's large size suggests that it may have developed several weeks before our evaluation. The presence of CH associated with thyroid enlargement in this patient is attributable to the mutation in the TG gene.

PS has an estimated prevalence ranging from 7.5 to 10 per 100,000 individuals [10, 11] and is classically defined by the combination of sensorineural deafness, goiter, and abnormal organification of iodide, with or without hypothyroidism.

As in our case, the SLC26A4 gene plays a crucial role in PS. Mutations in this gene are inherited in an autosomal recessive manner to develop the syndrome. The hallmark of the syndrome is the impaired hearing, which is associated with inner ear malformations such as an enlarged vestibular aqueduct. The thyroid phenotype is variable and goiter in PS may be present in 50%–83% of cases [28]. Goitrous enlargement can occur in varying degrees among patients with PS and can be progressive during childhood and adolescence, with the most significant enlargement often observed between the ages of 20 and 30 years [10, 11]. Fetal goiter is not a common clinical feature in PS, and in our case, the mutation in SLC26A4 seems to be more strongly linked to sensorineural deafness than to goiter. In terms of thyroid function, the majority of PS patients exhibit euthyroid goiter, however, certain patients exhibit signs of hypothyroidism [10, 11]. As in this case, PS frequently also causes structural abnormalities in the inner ear, such as an enlarged vestibular aqueduct or cochlear malformations, including mondini dysplasia, which can impair vestibular function and contribute to balance issues. Thus, in addition to managing thyroid conditions, hearing aids are recommended in PS, and in severe cases like ours, cochlear implants can be highly effective in improving hearing.

We were the first to report an association between the TG gene and PS in a patient with no family history of deafness or goiter. The combination of genetic factors suggests that the patient's condition may be due to mutations in multiple genes, each contributing to the clinical presentation.

This scenario could reflect a complex inheritance pattern, where mutations in different genes (such as TG and SLC26A4) are involved simultaneously. Although these genes are typically associated with distinct disorders, they may interact to create an overlapping or more severe phenotype. Specifically, the trans configuration of the mutations present in the consanguineous parents explains the patient's phenotype, characterized by fetal goiter with CH and sensorineural deafness, the latter of which is classifiable as PS. Functional analysis could provide further insight into the specific impact of the identified mutations.

In conclusion, concomitant mutations in the TG and SLC26A4 genes have not been previously described. The independent roles of each gene and their potential interaction may explain the clinical presentation of the patient. This rare genetic constellation highlights the importance of also considering multiple gene interactions when diagnosing and managing inherited disorders.

Funding Statement

This research received no external funding.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Ethics Statement

The study was conducted in accordance with the Declaration of Helsinki. Ethical review and approval were waived for this case report in accordance with local or national guidelines, as the case does not involve any intervention that deviates from standard practice and is not considered research.

Consent

Written informed consent has been obtained from the parents/to publish this article.

Disclosure

All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Conceptualization: Valeria Calcaterra and Mariano Lanna. Methodology: Valeria Calcaterra, Mariano Lanna, Elisa Ligato, Alberto Dolci, Arianna Laoreti, Irene Daniele, Elisa Cattaneo, Gianluca Lista, Valeria Savasi and, Gianvincenzo Zuccotti. Writing – original draft preparation: Valeria Calcaterra, Mariano Lanna, Elisa Ligato, Alberto Dolci, Arianna Laoreti, Irene Daniele, and Elisa Cattaneo. Writing – review and editing: Valeria Calcaterra, Mariano Lanna, Alberto Dolci, Irene Daniele, Elisa Cattaneo, Gianluca Lista, Valeria Savasi, and Gianvincenzo Zuccotti. Supervision: Valeria Calcaterra, Mariano Lanna, Gianluca Lista, Valeria Savasi, and Gianvincenzo Zuccotti. Valeria Calcaterra and Mariano Lanna contributed equally.