Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism

Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.


Introduction
Congenital primary hypothyroidism (CH; OMIM 218700) is the most common metabolic disorder in newborns with an estimated prevalence of 1 in 2500-3500 births [1,2]. It is characterized by an impaired neurodevelopment and can lead to intellectual disability and growth retardation if untreated. Early diagnosis and therapy are absolutely critical to avoid brain damage. If the serum fT4 concentration is below, and TSH clearly above, the age-specific reference interval, then levothyroxine (LT4) treatment should be started immediately [3].
A genetic origin of CH has been supported by various evidence ( [4]; recently reviewed by [2,5]). Familial forms of CH are, however, uncommon. Congenital hypothyroidism can be caused by thyroid dyshormonogenesis or thyroid dysgenesis. Only a minority of children with congenital hypothyroidism present thyroid dyshormonogenesis, while the majority (over 80%) present thyroid dysgenesis, a developmental anomaly of the thyroid. In thyroid dysgenesis, different developmental genes including PAX8, FOXE1, NKX2-1, NKX2-5, HHEX, and others have been shown to be affected and heterozygous dominant mutations prevail. All known genes together can only explain a low proportion of the cases [5].
To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn with high TSH levels together with his unaffected parents. A de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). Two further ZBTB26 gene variants were identified in a cohort of 156 individuals with congenital hypothyroidism. Pathway and network analysis indicated functional links of ZBTB26 to other known genes underlying thyroid dysgenesis. GWAS studies indicated a significant association with height. Additionally, we report that morpholino knock-down of zbtb26 in X. laevis led to smaller thyroid anlagen and that the Xenopus ortholog of human PAX8 could be rescued by zbtb26, thus giving functional support for the involvement of thyroid dysgenesis.

Exome Sequencing and Filtering
Sequencing was performed using the Illumina HiSeq 2000 system. Raw sequence data were mapped to the 1000 genomes phase II reference genome (GRCh37 version hs37d5) using BWA 0.6.2. Samples showed an average coverage of 94.92×, and >99% of bases in the autosomal chromosomes of the target regions have ≥ 10× coverage and ≥20 QUAL score. SNVs and indels with a read depth of at least 10× and minimum QUAL score of 20 were considered. Non-synonymous and splice site affecting SNVs and all exonic indels were filtered further. The genotype predicted by Platypus was used to assign genotypes to the variants. Minor allele frequency (MAF) from gnomAD genes and exomes was used and variants with MAF > 0.1% were removed. In total, 1198 exomes and 3910 WGS samples from the in-house database were used as control to remove common variants and variants present in more than 1% of the control samples.

Sanger Sequencing of Variants
Thirty-three variants were confirmed by Sanger sequencing. Polymerase chain reaction (PCR) with primers indicated in Supplementary Table S1 was performed with HotStar Taq Polymerase and products analyzed on agarose gels and subsequently sequenced using an ABI machine (Genewiz).

Network and Pathway Analysis by Ingenuity Software
QIAGEN Ingenuity Pathway Analysis (IPA; https://digitalinsights.qiagen.com/, accessed on 15 September 2021) was applied to predict functional connections, and their interpretation was carried out in the context of protein networks that comprise protein-protein interactions and related biological functions as well as canonical signaling pathways. ; and NIH Genotype-Tissue Expression (GTEx) project. Gene expression of ZBTB26 was accessed in 51 tissues by the NIH Genotype-Tissue Expression (GTEx) database, which is based on RNA-seq data from 8555 tissue samples obtained from 570 adult post-mortem individuals (V6, October 2015).

Experimental
Approaches with X. laevis X. laevis care and maintenance, morpholino design, and microinjections, as well as preparation of embryo sections, are given in detail in the Supplementary data.

In Situ Hybridization
A full-length zbtb26 RNA probe was used to perform whole-mount in situ hybridization (ISH). Fixation of embryos was done in MEMFA for 2-3 h at room temperature. ISH was performed with a Xenopus standard protocol [6]. RNA in situ probes were synthesized with SP6 or T7 polymerases. For tissue-specific analyses of mRNA enrichment after ISH, stained embryos were embedded in 4% formaldehyde for 3 h at room temperature. Embryos were embedded in a glutaraldehyde-crosslinked gelatin-albumin mix. Then sectioning was performed with a vibratome (Leica VT1000S; sections 30 µm).

Analyses of Thyroid Anlagen
Thyroid sizes were quantified at stage 45, when bilateral anlagen were still found in the ventral mesenchyme. Embryos were frontally sectioned and manually screened for thyroids. Equal settings were used to take pictures (10× objective of the same microscope, same filter set, same field of view, and same contrast and brightness adjustments). Embryos were only used when their anlagen (or the expected area with remnant tissue) were identified clearly. For individual embryos, the sections with the largest cross-sectional area of the thyroid anlagen were used for each slide [7]. The relative size of both thyroids was determined by measuring the surface area (square pixels) with the ImageJ polygon tool [8]. Then the obtained values were graphically represented as boxplots. Statistical calculations were performed using statistical R, with an unpaired Wilcoxon rank sum test with continuity correction (www.R-project.org, accessed on 15 September 2021).

Clinical Data
Patients included in this study were identified by the newborn screening at the University Children's Hospital Heidelberg. Screening of TSH concentration was measured in dry-blood spots and was carried out by the University of Heidelberg neonatal screening laboratory. Informed consent for exome or targeted sequencing was provided by the respective parents.
Patient 1 with a de novo L75S mutation in ZBTB26 is a eutrophic newborn Caucasian male with 3390 g birth weight, 56 cm birth length, and 37 cm head circumference. Congenital hypothyroidism was detected with a TSH level of 179 mU/L and normal values for T4, T3, and fT4. Due to the dramatically elevated TSH level, treatment was initiated. Therapy was started with levothyroxine at day 6 and normalization of TSH level was achieved at day 7. No thyroid autoantibodies were detected, and thyroglobulin was initially 159 ng/mL, indicating remaining thyroid tissue. Postnatal ultrasound detected thyroid tissue in the expected position, but no associated malformations. Ultrasound at the age of 9 years, however, detected no thyroid tissues and thyroglobulin was 8.7 ng/mL while TSH was elevated, indicating thyroid hypoplasia. The boy presented normal intelligence, but dyscalculia and dyslexia at school age. To maintain euthyroidism, increasing dosage of levothyroxine was necessary over the time. The family members were Caucasian and non-consanguineous. The grandfather suffered from thyroid nodules, but the younger sister was healthy with normal thyroid function.
Patient 2 with H236R mutation is a eutrophic newborn Caucasian female. Congenital hypothyroidism was detected with a TSH level of 59 mU/L and reduced values for T4, T3, and fT4. Therapy was started postnatally with levothyroxine, and normalization of TSH level was achieved. The parents reported that they were healthy, but no thyroid examination was performed and they were not available for DNA analysis.
Patient 3 with an intronic variant is a eutrophic Caucasian newborn male, born at 38 gestational weeks with 3500 g birth weight and 53 cm birth length. Congenital hypothyroidism was detected with a TSH level >50 mU/L and reduced values for T4, T3, and fT4. Therapy was started postnatally with levothyroxine and normalization of TSH level was achieved. Ultrasound at the age of 6 years detected absence of both thyroid and ectopic tissues. There was normal psychomotoric development. To maintain euthyroidism, increasing dosage was necessary over the time. Healthy parents were not available for analysis.
Another patient 332,756 was identified by DECIPHER and presented a 418 kb deletion encompassing ZBTB26 and 5 additional genes (RC3H2, ZBTB26, RABGAP1, GPR21, and STRBP). The patient had a pulmonary stenosis and hypothyroidism, as well as delayed speech and language development. This patient was not included in the analysis as the deleted region contained ZBTB26 as well as an additional 5 genes. Causality of ZBTB could therefore not shown.

Exome Sequencing, Filtering and Selection of Candidate Genes
To identify novel candidate genes implicated in the etiology of congenital hypothyroidism, exome sequencing has been carried out in an affected child and his unaffected parents. Raw sequence data were mapped to the 1000 genomes phase II reference genome (hs37d5) followed by a bioinformatical variant detection pipeline (see Material and Methods). After specific filtering, 650 candidate genes (SNV or Indels) were identified. Further filtering excluded those genes with a minor allele frequency above 0.01 in gnomAD, a consensus score above 1 and a negative intolerance score. Variants were then analyzed for their effect on protein function if they had been predicted as damaging by at least one of the prediction tools (Mutation Taster, Polyphen2, PROVEAN, and SIFT), and with a CADD above 13. A CADD score above 13 indicates that the variant is within the 5% of the most deleterious substitutions in the human genome. De novo variants were prioritized.
A de novo variant c.224A>G with the top CADD score of 25.4 was identified in the ZBTB26 gene, leading to the amino acid exchange p.L75S ( Figure 1A). ZBTB26 (Zink finger and BTB domain -containing protein 26) is a DNA-binding protein regulating transcription. It is a small, highly conserved gene with a transcript conservation between human and Xenopus of 73% identity. Somatic mutations in ZBTB26 have been identified in different cancers including thyroid carcinoma (https://cancer.sanger.ac.uk/cosmic/gene/analysis? ln=ZBTB26, accessed on 15 September 2021). ZBTB26 consists of two exons (the first exon is not translated) and a transcript length of 4455 base pairs, encoding a protein of 441 amino acids. The affected Leucine (L) in this individual is highly conserved in mice (M. musculus), rats (R. norvegicus), dogs (Canis), cows (B. taurus), chickens (G. gallus), and frogs (Xenopus), suggesting functional significance. The amino acid change was predicted to be disease-causing/damaging by all the used prediction programs and had previously not been identified in gnomAD (Supplementary Table S1).

Cohort Screening of 156 Children with Congenital Hypothyroidism for ZBTB26 Variants
To find out how frequent variants in the ZBTB26 gene are in children with primary hypothyroidism, Sanger Sequence analysis was carried out in an additional cohort of 156 patients with congenital hypothyroidism due to thyroid dysgenesis. Variants in two further individuals were identified, one with a c.707T>C substitution, causing an amino acid exchange p.H236R, and another with an intronic variant, 58 base pairs before the exonintron junction. DNA of the parents of both patients was not available for analysis. Both variants were predicted to have a CADD score of 17.8 and 17.6. The amino acid change affected a highly conserved amino acid (Histidine 236) between humans and mice (M. musculus), rats (R. norvegicus), dogs (Canis), cows (B. taurus) and chickens (G. gallus). In gnomAD, it was detected in 4 out of 141,406 Europeans, resulting in an allele frequency of 0.00003098. The variant was completely absent in East Asians, but present in 487 Africans, indicating differences among populations. In summary, 2 out of 156 individuals with congenital hypothyroidism presented a risk variant of unknown functional significance in the ZBTB26 gene (Supplementary Table S1).

Network Analysis
To find out if ZBTB26 shares any common functional links with the known genes involved in primary hypothyroidism (indicated in blue color on Figure 1), Ingenuity pathway analysis was carried out. Network links between ZBTB26, UBE2I, NKX2-1, and PAX8 could be revealed; all reside in the nucleus ( Figure 1B). Protein-protein interactions were

Cohort Screening of 156 Children with Congenital Hypothyroidism for ZBTB26 Variants
To find out how frequent variants in the ZBTB26 gene are in children with primary hypothyroidism, Sanger Sequence analysis was carried out in an additional cohort of 156 patients with congenital hypothyroidism due to thyroid dysgenesis. Variants in two further individuals were identified, one with a c.707T>C substitution, causing an amino acid exchange p.H236R, and another with an intronic variant, 58 base pairs before the exon-intron junction. DNA of the parents of both patients was not available for analysis. Both variants were predicted to have a CADD score of 17.8 and 17.6. The amino acid change affected a highly conserved amino acid (Histidine 236) between humans and mice (M. musculus), rats (R. norvegicus), dogs (Canis), cows (B. taurus) and chickens (G. gallus). In gnomAD, it was detected in 4 out of 141,406 Europeans, resulting in an allele frequency of 0.00003098. The variant was completely absent in East Asians, but present in 487 Africans, indicating differences among populations. In summary, 2 out of 156 individuals with congenital hypothyroidism presented a risk variant of unknown functional significance in the ZBTB26 gene (Supplementary Table S1).

Network Analysis
To find out if ZBTB26 shares any common functional links with the known genes involved in primary hypothyroidism (indicated in blue color on Figure 1), Ingenuity pathway analysis was carried out. Network links between ZBTB26, UBE2I, NKX2-1, and PAX8 could be revealed; all reside in the nucleus ( Figure 1B). Protein-protein interactions were identified between ZBTB26 and UBE2I, as well as UBE2I and NKX2-1 [9], and an interaction between PAX8 and NKX2-1 has been described [10]. Together, these data highlight a connection between ZBTB26 and several known CH genes ( Figure 1B).

GWAS and Disease Knowledge Databases
To predict the pathological relevance of the identified ZBTB26 gene in a human phenotype, we consulted the Disease Knowledge Portal (https://cvd.hugeamp.org, accessed on 15 September 2021) and found GWAS associations of genome-wide significance of several intron variants of CYP26B26 with height

Experimental Data in X. laevis Provides Functional Support
As ZBTB26 is expressed in human thyroid gland according to GTEx (among other tissues), we functionally assessed ZBTB26 in thyroid development using the African clawed frog X. laevis as an animal model. Expression of the orthologous zbtb26 gene in the thyroid anlage was shown by in situ hybridization analyses at different stages of tadpole development (tailbud stage 32 to tadpole stage 45). Strong signals in the ventral foregut area (Figure 2A,B) and in the thyroid precursor were detected (Figure 2A',B'). This was also the case in older tadpole stages, when thyroid anlagen are already split and relocated medially, as well as in the mouth floor epithelium ( Figure S1A,B).
Mid-sagittal sections showed specific reduction in pax2 in the pre-thyroid tissues of Zbtb26 knockdowns compared to control siblings ( Figure 2C',D'). Further supporting a functional interaction of both genes, sections through the thyroid anlagen of a stage 37 embryo revealed overlapping expression in this area ( Figure S1D,E). The effect on pax2 was specific, as reintroduction of wildtype Xenopus zbtb26 lacking the MO-binding sequence in the 5 UTR rescued the pax2 expression in a significant manner. Interestingly, injecting zbtb26 mRNA alone seemed to have an enhancing or stabilizing effect on pax2 expression as well ( Figure 2E).
Next, we wanted to analyze if this reduction in pax2 expression after zbtb26 loss of function also correlates with a change in thyroid tissue development in early tadpoles. We performed knockdown experiments, either targeting both anlagen or injecting unilaterally into the right or left lineage to hit only one half of the embryo (strictly separated in Xenopus early development), then used the opposite halves as internal controls. When analyzing tadpole frontal sections through the already separated thyroid anlagen at stage 45, we found most morphant thyroids to be reduced in size or not developed at all (Figure 2F-H). Quantification of bilaterally injected specimens revealed an average reduction in size by 36% in morphant embryos ( Figure 2I). Similarly, specimens targeted unilaterally with the zbtb26-MO showed about 28% smaller thyroid anlagen on the injected side than those of the non-injected endogenous control halves ( Figure S1F). These results support the conclusion that zbtb26 expressed in the thyroid precursor tissue is required for normal thyroid development.

Discussion
Since the identification of the first causal gene for thyroid dysgenesis PAX8 in 1998 [18], a number of genes underlying congenital primary hypothyroidism (CH) have been reported to play a role in this phenotype (e.g., TSHR, NKX2-1, NKX2-5, FOXE1, GLIS3, JAG1, TBX1, NTN1, CDCA8, HOXD3, HOXB3, CDCH8, TUBB1, and TRPC4AP) [5,19,20]. These advances have contributed to our understanding of thyroid development and provided very useful information for genetic diagnosis. However, the underlying molecular mechanisms are still poorly understood, and the vast majority of patients and their families are still lacking a genetic diagnosis.
We have provided genetic and functional evidence that ZBTB26 is a further risk gene for this disorder. The ZBTB26 gene is a member of the large ZBTB family of proteins, which are highly conserved transcription factors with essential functions during development (Cheng et al., 2021). All ZBTB family members contain a BTB protein binding and Kruppeltype zink finger DNA binding domain. The BTB domain is used for protein-protein interactions, while the zinc finger domain primarily functions as a DNA binding module. The precise role of most ZBTB family members is still unknown. Several ZBTB proteins have emerged as critical factors during development which regulate the lineage commitment, differentiation and function of lymphoid cells as well as many other developmental processes [21].
In this study, we have identified a de novo missense in ZBTB26 detected in a child with CH, which was not present in his unaffected parents. This child, as well as the other patients analyzed, were all derived from the German newborn screening program. Two additional variants in ZBTB26 were identified in the cohort of 156 children with CH, but as the parents were not available in this study, it remains unsolved if these variants occurred de novo or not. As the detected ZBTB26 variants were rare in this cohort (2/156), a high genetic heterogeneity is presumed, which is in line with former investigations.
An oligogenic model of inheritance for CH has also been proposed by previous studies, possibly together with a modulation by environmental modifiers [5,22]. This may also apply to our patients, at least to the patient, where exon sequencing has been performed. In this patient with the de novo ZBTB26 mutation, further weak variants in additional known CH genes (PAX8, TUBB1) were transmitted from either parent to the child (the PAX8 variant from mother; the TUBB1 variant from father), suggesting that a combination of different variants would be needed to lead to the full-blown phenotype (Supplementary Table S2). The co-occurrence of de novo and familiar mutations in genes involved in CH also highlight that epistasis between different specific gene loci can modulate the risk for CH. Different susceptibility variants for CH variants may thus act together, and in some cases, additional epigenetic or environmental factors may be also necessary for the emergence of the disorder. This oligogenic model of inheritance or multi-hit model for CH has been already proposed before [5,22] and may also apply to our patients.
Finally, functional studies have shown that ZBTB26 represents a convincing risk gene for CH. Network analysis has shown that ZBTB26 is linked to several known CH genes (CDCA8, NKX2-1, and IKBKG) via UBE2I, and according to the GTEX database, ZBTB26 has one of its highest expressions in the thyroid. In Xenopus, which was used as an animal model, zbtb26 transcripts were found in the early thyroid anlagen and continued to be active in early thyroids at different stages of tadpole development (Figures 2 and S1A,B). Gene activity can easily be manipulated in Xenopus by microinjection into its large early embryos right after fertilization. These knockdown experiments demonstrated significantly smaller thyroid anlagen and smaller thyroids at the tadpole stage, thus indicating a direct effect of its reduced expression.
In summary, a causal link of ZBTB26 variants and CH is supported by several lines of evidence: Variants with evolutionary conservation of affected amino acids and good algorithm predictions with an effect on protein function and low frequency in the general Caucasian population were detected in altogether 3/157 individuals with CH. In addition, reported associations with final height and the gene expression patterns in the thyroid in human and frog supported these findings. Finally, the regulatory role on pax2 expression, and the in vivo requirement for early thyroid organogenesis in tadpole furthermore speak for a relevance of this gene in CH. Our study also provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH. To date, it is not clear how many genes contribute to the phenotype of CH and how these different variants interact with each other. A better knowledge of these complex interactions will be needed to fully understand this common endocrine disorder.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/genes12121862/s1: Table S1: Identified ZBTB variants in patients with congenital hypothyroidism, Table S2: Additional variants in known hypothyroid genes and gnomAD analysis of variants compared with ZBTB26, Figure S1: zbtb26 has a conserved role in amphibian thyroid development; Supplementary data: Xenopus laevis: care and maintenance, morpholino design, microinjection and embryo sections.