TYPE Case Report PUBLISHED 11 October 2022 DOI 10.3389/fendo.2022.957969 Novel variant in HHAT as a OPEN ACCESS cause of different sex EDITED BY Amit V. Pandey, University of Bern, Switzerland development with partial REVIEWED BY Hiroshi Kurosaka, gonadal dysgenesis associated Osaka University, Japan Sorahia Domenice, Clinical Hospital, University of with microcephaly, eye defects, São Paulo, Brazil *CORRESPONDENCE and distal phalangeal hypoplasia Núria Camats-Tarruella nuria.camats@vhir.org of both thumbs: Case report † These authors share first authorship SPECIALTY SECTION Noelia Baz-Redón 1,2†, Laura Soler-Colomer 3†, This article was submitted to Pediatric Endocrinology, Mónica Fernández-Cancio 1,4, Sara Benito-Sanz 4,5, a section of the journal Frontiers in Endocrinology Marta Garrido 6, Teresa Moliné 6, Marı́a Clemente 1,2,3,4, RECEIVED 31 May 2022 Núria Camats-Tarruella 1,4* and Diego Yeste 1,2,3,4 ACCEPTED 15 September 2022 1 Growth and Development Group, Vall d’Hebron Research Institute (VHIR), Hospital Universitari Vall PUBLISHED 11 October 2022 d’Hebron, Barcelona, Spain, 2 Pediatrics, Obstetrics and Gynecology and Preventive Medicine CITATION Department, Universitat Autònoma de Barcelona, Barcelona, Spain, 3Pediatric Endocrinology Section, Baz-Redón N, Soler-Colomer L, Hospital Universitari Vall d’Hebron, Barcelona, Spain, 4Centre for Biomedical Network Research on Fernández-Cancio M, Benito-Sanz S, Rare Diseases (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain, 5Institute of Medical and Garrido M, Moliné T, Clemente M, Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autonóma de Madrid, Camats-Tarruella N and Yeste D Madrid, Spain, 6Department of Pathology, Hospital Universitari Vall d’Hebron, Barcelona, Spain (2022) Novel variant in HHAT as a cause of different sex development with partial gonadal dysgenesis associated with microcephaly, eye defects, and distal phalangeal The palmitoylation of the Hedgehog (Hh) family of morphogens, named sonic hypoplasia of both thumbs: hedgehog (SHH), desert hedgehog (DHH), and Indian hedgehog (IHH), is Case report. Front. Endocrinol. 13:957969. crucial for effective short- and long-range signaling. The hedgehog doi: 10.3389/fendo.2022.957969 acyltransferase (HHAT) attaches the palmitate molecule to the Hh; therefore, COPYRIGHT variants in HHAT cause a broad spectrum of phenotypes. A missense HHAT © 2022 Baz-Redón, Soler-Colomer, novel variant c.1001T>A/p.(Met334Lys) was described in a patient first referred Fernández-Cancio, Benito-Sanz, Garrido, Moliné, Clemente, for a 46,XY different sexual development with partial gonadal dysgenesis but Camats-Tarruella and Yeste. This is an with microcephaly, eye defects, and distal phalangeal hypoplasia of both open-access article distributed under thumbs. The in silico analysis of the variant predicted an affectation of the the terms of the Creative Commons Attribution License (CC BY). The use, nearest splicing site. Thus, in vitro minigene studies were carried out, which distribution or reproduction in other demonstrated that the variant does not affect the splicing. Subsequent protein forums is permitted, provided the in silico studies supported the pathogenicity of the variant, and, in conclusion, original author(s) and the copyright owner(s) are credited and that the this was considered the cause of the patient’s phenotype. original publication in this journal is cited, in accordance with accepted KEYWORDS academic practice. No use, distribution or reproduction is 46,XY different sexual development, syndromic DSD, HHAT, minigene studies, permitted which does not comply with case report these terms. Frontiers in Endocrinology 01 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 Introduction acyltransferase (MBOAT) domain (amino acids 150–442) (Figure 1) (1, 8, 9). This palmitoylation is critical for effective The Hedgehog (Hh) family consists of morphogens short- and long-range signaling of Hh molecules (8). implicated in the control of cell growth, survival, HHAT gene (MIM: 605743) contains 12 exons, with its differentiation, and embryonic pattern for a variety of organs coding DNA sequence (CDS) extending from exon 2 to 12. and tissues (1). The three Hh ligands are sonic hedgehog (SHH), Genetic variants in this gene have been related to Nivelon– desert hedgehog (DHH), and Indian hedgehog (IHH) (1). SHH Nivelon–Mabille syndrome with phenotypes similar to the loss is expressed in early embryogenesis and acts in neural, cranial, of Hh function (Figure 1) (10). To date, and according to our skeletal, and limb patterning. Later in development, during knowledge, a total of 10 HHAT variants have been described in organogenesis, SHH is expressed in most epithelial tissues and the literature, six of them missense, three gross deletions, and is involved in the development of lungs, kidneys, hair, teeth, and one nonsense. The described deletions were associated with eyes (1). Loss of SHH expression and function causes cyclopia schizophrenia (11), ankylosing spondylitis (12), and persistent and defects in the patterning of the ventral neural tube, somites, cloaca (13). Regarding the nonsense variant, it was described in a and foregut. Also, later defects include severe distal limb case with pituitary stalk interruption syndrome (PSIS) (14). malformation, absence of vertebrae and most of the ribs, and Conversely, the missense variants were reported in patients failure in lung branching (1). DHH expression is largely with a broad spectrum of phenotypes according to the restricted to gonads, so it is crucial for testis and ovarian impairment of SHH (i.e., intellectual disability, eye development and function (1, 2). It is secreted by Sertoli cells abnormalities, or limb malformations), DHH (DSD), and/or and induces the formation of the Leydig cell lineage (3). Genetic IHH (i.e., dwarfism or brachydactyly) (Figure 1) (7, 15–19). variants in DHH cause 46,XY different sexual development We present an interesting case of a patient with a HHAT (DSD) with partial or complete gonadal dysgenesis (2–6). IHH novel variant first referred for DSD but with a later diagnosis of is specifically expressed in a limited number of tissues and acts affectation in the other Hh pathways. Moreover, the candidate on bone growth and pancreas development. Defects in IHH gene HHAT variant was an exonic variant with a predicted splicing reduce chondrocyte proliferation causing bone growth defects effect, so it was studied in vitro. such as dwarfism, acrocapitofemoral dysplasia, or brachydactyly (1, 7). Hh proteins undergo multiple processing steps, during their passage through the secretory pathway, required for the Case report generation and release of the active functional ligand. After the signal sequence is removed, the Hh proteins suffer an The patient is a 13-year-old Pakistani girl who was admitted autoproteolysis followed by a linkage of a cholesterol molecule to the pediatric endocrinology outpatient clinic for hirsutism to the C-terminus. Subsequently, a molecule of palmitate is and lack of breast development. She was born by normal vaginal covalently attached to the N-terminus of Hh by the hedgehog delivery at 38 weeks of gestation with a birth weight of 2,025 g acyltransferase (HHAT). HHAT is a protein with 10 (−2.5 SDS for gestational age) after an uneventful pregnancy. transmembrane domains and a catalytic membrane-bound O- Her parents were first cousins, and she has two younger FIGURE 1 Diagram of HHAT gene and protein. Diagram of HHAT gene (NC_000001.11) with the boxes indicating the 12 exons (e1 to e12). The coding DNA sequence (NM_018194) from e2 to e12 codifies for HHAT protein (striped lines). Diagram of the HHAT protein isoform NP_060664.2 with its different domains and localization of the previously described variants in literature. The membrane-bound O-acyltransferase (MBOAT) domain encompasses amino acids 150 to 442. Frontiers in Endocrinology 02 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 unaffected female siblings. No remarkable familial medical (large diameter in right eye), a retinal pigment epithelium history was reported. Due to microcephaly, a brain magnetic atrophy, a temporary flat major excavation in the right eye resonance imaging (MRI), performed at the age of 7, showed applied retina, and normal peripheral vision in both eyes. biparietal gliosis and necrosis caused by a hypoxic–ischemic After 6-month-long psychological counseling, in which the perinatal injury. She does not suffer from an intellectual patient confirmed a female gender identity, a laparoscopic disability. When she was almost 8 years old, she was gonadectomy was performed. The vaginoscopy showed a diagnosed with different sexual development, with hypoplastic vagina terminated in a blind pouch of about 7.5 cm in length, majora and minora labia, and a microphallus (length and width with the absence of the cervix and uterus. Both testes were small, unavailable). No other clinical signs of hyperandrogenism were each one measuring around 1.5 × 1 × 0.8 cm. Microscopically, detected. Her external genital score (EGS) was 0 according to both dysgenetic gonads contained immature seminiferous van der Straaten et al. (20) and Ahmed and Rodie (21). tubules, 70% of which had germinal cells, and Leydig cell Laboratory hormonal assays revealed high total testosterone hyperplasia was observed (Figure 2C, left and magnified levels of 20 ng/dl (normal reference range for prepubertal female images). In the central zone of the right testis parenchyma, a <10 ng/dl) and normal levels of 17-hydroxyprogesterone at 0.52 tumor cell proliferation of 5 mm was identified (Figure 2C, right ng/ml (normal reference range <1 ng/ml). image). Immunohistochemically, the tumor cells were positive Physical examination showed that the patient had external for placental alkaline phosphatase (PLAP), cKIT, D2-40, and genitalia virilization, with an increased amount of pubic hair OCT4 and negative for AFP. No vascular, lymphatic, or extended to her inner thigh area, microphallus, and hypoplastic spermatic cord invasion was observed. The tumor was labia. Her urethral meatus was located on the ventral side of the classified as a germinoma. No neoplastic infiltration was phallus. She had no palpable gonads, and a vaginal introitus was identified in the left testis. Four months after the present. Her shoulders were relatively broad, and no breast gonadectomy, the phallus started to involute, and a feminizing development was observed. Her voice was deepened, and her hormonal treatment of 2 µg/day of ethinylestradiol was started. hirsutism, graded with a modified Ferriman and Gallwey score, was severe. The patient’s weight was 42.5 kg (−0.8 SDS), and her height was 162.8 cm (+1 SDS). She had no abnormal Molecular analysis dysmorphic features. Laboratory assays revealed the following hormonal values: Genomic DNA was isolated from whole peripheral blood luteinizing hormone (LH) 23.9 IU/L, follicle-stimulating samples from the patient and the parents using standard hormone (FSH) 103.5 IU/L, estradiol 25.5 pg/ml, testosterone procedures. DNA samples were analyzed with a custom- 325.6 ng/ml, dihydrotestosterone 280 pg/ml, androstenedione designed high-throughput panel with genes related to DSD 2.2 ng/ml, dehydroepiandrosterone sulfate (DHEAS) 181.48 mg/ (DSDSeq.V1, 112 genes and three regulatory regions) using dl, 17-hydroxyprogesterone 1.26 ng/ml, cortisol 22.8 mg/dl, anti- SeqCap EZ technology (Roche Nimblegen) as previously Müllerian hormone (AMH) 0.45 ng/ml, inhibin B 21.6 pg/ml, described (23). The genetic result was confirmed by Sanger and prolactin 32.3 ng/ml. Her karyotype was 46,XY. The sequencing. A novel homozygote variant in HHAT was abdominopelvic ultrasound showed the absence of a uterus identified (MIM: 605743) in the patient, and both parents and ovaries, a filiform vagina up to its lower third, and two were carriers. No further variants of interest were identified oval-shaped structures located in the upper third of the inguinal using the DSDSeq.V1 NGS panel. The HHAT (NM_018194) canal, probably corresponding to degenerated testes. These variant c.1001T>A/p.(Met334Lys), located at the end of exon 8, structures presented microcalcifications and vascularization, was classified as a variant of uncertain significance (VUS) and the one on the left side had millimetric hypoechoic according to the American College of Medical Genetics and lesions, which can be considered suspicious for malignancy. A Genomics (ACMG) criteria (PM2 and PP3) (Figure 2D). This pelvic MRI confirmed these findings, showing bilateral vas variant is located close to the exonic end, and possible variant deferens and seminal vesicles. Tumor markers (a-fetoprotein splicing effects were analyzed with the help of Alamut v2.11 (AFP), human chorionic gonadotropin, and carcinoembryonic splicing tools (SpliceSiteFinder, MaxEntScan, NNSPLICE, antigen) were negative. All these findings are concordant with a GeneSplicer, and Human Splicing Finder), which predicted a DSD with partial gonadal dysgenesis. possible effect at the nearest splice site. The bone age (13 years of chronological age) was determined to be 15 years by the Tanner–Whitehouse method. The skeletal survey at 13.5 years of age revealed microcephaly, with a head Minigene in vitro studies circumference of 51.8 cm (SD −2.65) (22) (Figure 2A) and hypoplasia of distal phalanges of both thumbs and bilateral RNA sequencing studies were performed with the aim of capito-hamate coalition (Figure 2B). The ophthalmologic confirming the in silico software prediction that this variant evaluation showed an eye fundus with asymmetric papilla likely affected the nearest splice site. HHAT RNA could not be Frontiers in Endocrinology 03 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 A B C D FIGURE 2 Physical examination, clinical signs, and HHAT candidate variant in our 46,XY patient. (A) Microcephaly visible in X-rays. (B) Skeletal survey showed hypoplasia of distal phalanges of both thumbs and bilateral capito-hamate coalition. (C) The anatomopathological analysis of the right and left dysgenetic gonads showed immature seminiferous tubules (ST) with hyperplasia of Leydig cells (LC) (left and magnified image) and a germ-cell (positive for placental alkaline phosphatase (PLAP) staining) tumor in the right gonad (asterisk) (right). (D) Family pedigree (right) and electropherogram (left) showing the proband (II-1, arrow) and parental (I-1 and I-2) genomic DNA with a T>A substitution observed in the 1001 position of HHAT gene (NM_018194). Both parents were carriers. isolated from peripheral blood or saliva, so a minigene in vitro well plate and transiently transfected with the WT, mutant, or study for the HHAT variant c.1001T>A was performed. The both WT and mutant (heterozygous state) minigenes (250 ng/ wild-type (WT) minigene was obtained by PCR from genomic well) using Lipofectamine 2000™ (Invitrogen, Thermo Fisher DNA (gDNA) of human whole blood. This minigene was Scientific , Waltham, MA, USA) and following the designed to include exons 8 to 10 and ≥200 bp of the flanking manufacturer’s recommendations. Forty-eight hours after introns regions following the Riedmayr et al. protocol (24). The transfection, cells were washed with phosphate-buffered saline WT PCR-amplified product was cloned into a pcDNA3 vector (PBS) and frozen. RNA was lysed with TRIZOL reagent (Invitrogen by Life Technologies, Carlsbad, CA, USA). The (Invitrogen, Life Technologies), extracted following an in- c.1001A mutant minigene (MT) was generated by site-directed house protocol, and treated with DNase (Promega, Madison, mutagenesis following the QuickChange protocol (Agilent WI, USA). cDNA synthesis was performed by reverse Technologies Inc., Santa Clara, CA, USA) and the WT vector transcription (RT)–PCR (GoScript Reverse Transcription as a template. Both vectors were verified by Sanger sequencing. System, Promega). Finally, WT and mutant HHAT cDNAs For in vitro expression studies, COS-7 cells were grown in a 24- were amplified using the cloning primers, loaded in a 2% Frontiers in Endocrinology 04 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 agarose gel electrophoresis, and sequenced. All minigene MBOAT domain (Figure 4A). This domain is important for the transfections (WT/WT, WT/MT, and MT/MT) apparently main activity of the protein, and a residue change might disturb resulted in cDNA amplification products of the same size, this function. Regarding amino acid properties, there is a close to the length of minigene design including exons 8 to 10, difference in charge, size, and hydrophobicity between the corresponding to 416 bp (Figure 3A). The sequence analysis of wild-type and the mutant amino acid, because this genetic these products confirmed that all of them conserved the entire variant causes a change from a hydrophobic amino acid to a sequence of exons 8, 9, and 10 (Figure 3B), which means that the positively charged one. As the mutant amino acid introduces a variant does not affect splicing and not confirming the in silico charge, this can cause the repulsion of ligands or other residues prediction of aberrant splicing. with the same charge. Furthermore, as the mutant residue is bigger, this might lead to bumps. Finally, hydrophobic interactions, either in the core of the protein or on the surface, In silico studies may be lost. This residue is highly conserved throughout evolution (GERP 5.5599), except for Xenopus tropicalis, which As the effect on splicing was not confirmed, this candidate presents isoleucine, also a hydrophobic amino acid, in this variant was studied in silico, analyzing the specific location of the position. The conservation across species suggests an protein change using RCSB PDB (https://www.rcsb.org/), amino important role of this amino acid in the HHAT protein acid conservation through evolution by Clustal Omega (https:// structure, stability, or function (Figure 4A). Visualizing the www.ebi.ac.uk/Tools/msa/clustalo/), and the prediction of HHAT PDB model 7MHY (25), it can be observed that the protein stability changes caused by the variant using the I- protein residue Methionine 334 is one of the nearest (5 Å) amino Mutant2.0 website tool (https://folding.biofold.org/i-mutant/i- acids to the palmitate donor palmitoyl-CoA (Figures 4B–D). mutant2.0.html). The online tool HOPE (https://www3.cmbi. Moreover, this single-point variant was predicted to decrease umcn.nl/hope/method/) was used for further in silico analyses. HHAT protein stability, in terms of sequence and protein The HHAT variant p.(Met334Lys) is located in the functional structure using the same PDB model 7MHY (25). A B FIGURE 3 Results of the HHAT in vitro minigene functional studies. (A) Results of the HHAT in vitro minigene experiments. COS-7 cells were transfected with wild-type (WT) HHAT, c.1001A mutant HHAT (MT), or both WT/VA HHAT. RNA was extracted, and RT-PCR for HHAT cDNA was performed. The figure shows the PCR products on an agarose gel. (B) Electropherograms of the minigene PCR products. The arrow indicates the position of the nucleotide variant c.1001A. Hom, homozygous state; Het, heterozygous state. Frontiers in Endocrinology 05 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 A B C D FIGURE 4 HHAT p.(Met334Lys) variant in silico studies. (A) Diagram of the HHAT protein isoform NP_060664.2 with its different domains and localization of variant p.(Met334Lys) in membrane-bound O-acyltransferase (MBOAT) functional domain of the protein. The in silico studies using Clustal Omega showed that the mutated Methionine 334 is conserved through vertebrates. (B) 3D image of the HHAT molecule and the ligand palmitoyl-CoA (in pink) indicating the position of Met334 (PBD 7MHY, RCSB PDB). (C) 3D magnified image indicating that the 334 position is very close to the ligand palmitoyl-CoA. (D) HHAT peptide sequence showing the amino acids in the active center (<8 Å from the ligand) (in red), which includes Met334 (underlined). Discussion different mixed clinical manifestations (Figure 1). Callier et al. (7) described a familial case of two siblings with complex We describe a case of a homozygous patient for a novel phenotypes including 46,XY DSD, chondrodysplasia, and variant c.1001T>A/p.(Met334Lys) in HHAT using a custom congenital anomalies (Nivelon–Nivelon–Mabille syndrome). high-throughput DSD gene panel. HHAT gene encodes the The genetic analysis described a HHAT homozygous missense hedgehog acyltransferase HHAT, which catalyzes the variant (c.860G>T/p.Gly287Val) in the MBOAT domain palmitoylation of the Hh family SHH, DHH, and IHH (1, 8, (Figure 1) (7). Abdel-Salam et al. (16) described two sisters of 9). Therefore, variants in HHAT cause a broad range of a consanguineous family that presented with microcephaly and phenotypes consistent with the defects in Hh pathways (7, 11– cerebellar vermis hypoplasia. One of the sisters presented with 19). The patients of the described familial cases with brachydactyly with dysplastic nails, and the other one, optic homozygous missense HHAT variants also presented with atrophy. There was no evidence of dwarfism or gonadal Frontiers in Endocrinology 06 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 dysgenesis. An exome sequencing analysis identified a this candidate variant detected in the patient does not affect the homozygous variant in the MBOAT HHAT domain (c.770T>C/ splicing, contrary to the prediction (Figure 3). Considering these p.Leu257Pro) (Figure 1) (16). In a recent publication, Mazen et al. results, we demonstrate the importance of functional studies in (19) described a patient with 46,XY gonadal dysgenesis, bilateral order to confirm or refute bioinformatics predictions of inguinal testes with no Müllerian derivatives, and strong male variant pathogenicity. identity with microcephaly and multiple café au lait patches. A Having concluded that this variant does not affect RNA splicing, novel homozygous HHAT missense variant (c.1329C>A/ we decided to perform in silico studies to analyze its pathogenicity. p.Asp443Lys) was described as the cause of the patient’s phenotype The variant is located at the end of exon 8, causing the peptide (Figure 1) (19). sequence to change from Methionine to Lysine in 334 positions. It is In the particular case of the presented patient, although she located in the highly conserved MBOAT functional domain was firstly referred for DSD with gonadal dysgenesis, extended (Figure 4). From the previously described variants in HHAT, medical examination confirmed microcephaly with biparietal those that cause a phenotype similar to that of our patient are also gliosis, necrosis, and eye abnormalities (Figure 2). Considering located in MBOAT. The affected amino acid is highly conserved the role of Hh pathways and their defects, the clinical through evolution (Figure 4) and is near the ligand palmitoyl-CoA, manifestations of our patient are highly consistent with the and the variant seems to affect HHAT protein stability. All these alteration of SHH and DHH. The patient also presented with results lead us to think that the variant does affect the protein distal phalangeal hypoplasia of both thumbs (Figure 2B), which function and, therefore, might be the cause of the patient’s could be closely related to IHH defects. This is concordant with phenotype. However, as genetic variants in HHAT cause a broad mild skeletal defects caused by heterozygous IHH variants in spectrum of phenotypes and high variability in their severity, it recently published cases (26). However, previously reported IHH would be interesting to conduct functional studies to confirm the cell variants cause more marked defects such as severe short stature, downstream signaling defects resulting from genetic variants in brachydactyly, and severe shortening of the middle phalanges HHAT and improve understanding of Hh pathways. (27, 28). The previously described cases with homozygous In conclusion, we described a novel HHAT variant in a variants in HHAT showed phenotypes with clinical signs patient with DSD with partial gonadal dysgenesis, microcephaly, associated with the SHH pathway along with defects in DHH eye defects, and hypoplasia of the distal phalanges of both and/or IHH, concordant with our case. thumbs. This candidate variant was initially predicted to affect The microscopic analysis of the dysgenetic gonads of our splicing, but in vitro studies refuted this. As the in silico studies patient showed separated immature seminiferous tubules due to supported the pathogenicity of the variant, we considered it to be hyperplasia of Leydig cells (Figure 2C). Previous studies on Hhat the cause of the patient’s phenotype. loss of function in mice confirmed that Hhat is crucial for proper testis cord formation and differentiation of fetal Leydig cells so that there is a nearly complete absence of Leydig cells in mutant Data availability statement fetal testes (7). In the case of adult mutant testes, a reduced number of Leydig cells compared to normal adult testis with a The datasets for this article are not publicly available due to reminiscent expression of Leydig cell-specific marker CYP11A1 concerns regarding participant/patient anonymity. Requests to has been described (7). These results are not concordant with access the datasets should be directed to the corresponding author. ours, as we observed hyperplasia of this cell type in adult testis. Further studies are required to understand the precise function of HHAT in the differentiation of Leydig cells. Ethics statement The described missense variant in this patient c.1001T>A/ The studies involving human participants were reviewed and p.(Met334Lys) was located at the end of exon 8. Missense variants approved by CEIC Hospital Universitari Vall d’Hebron. Written are usually evaluated focusing on the amino acid change and not informed consent to participate in this study was provided by the on the nucleotide variant, but it has been demonstrated that participants’ legal guardian/next of kin.Written informed exonic variants might affect splicing. Based on in silico studies, it consent was obtained from the minor(s)’ legal guardian/next has been reported that up to 25% of the previously described of kin for the publication of any potentially identifiable images or variants classified as missense and nonsense may affect splicing data included in this article. (29). Therefore, it is highly recommended to use predictive tools for identifying splice variants. In our specific case, the bioinformatics splicing predictor software predicted that the Author contributions HHAT variant c.1001T>A affected the nearest splicing site. For that reason, in vitro minigene studies were carried out, which NB-R and LS-C wrote the manuscript. NB-R and NC-T showed that exons 8 to 10 were present in the WT and mutant contributed to the conception and design of the study, and minigenes (in homozygous and heterozygous states). Therefore, performed experimental studies. MF-C and SB-S performed Frontiers in Endocrinology 07 frontiersin.org Baz-Redón et al. 10.3389/fendo.2022.957969 genetic studies. MG and TM performed the histological analyses. Conflict of interest DY, MC and MG contributed to the diagnosis of the patient. All authors contributed to manuscript revision, read, and approved The authors declare that the research was conducted in the the submitted version. DY and NC-T supervised the project. absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This study was partly supported by a grant from the Fondo Publisher’s note de Investigació n Sanitaria (PI15/01647 [to MF-C and SB-S]). All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Acknowledgments organizations, or those of the publisher, the editors and the We thank Dr. Josep Castellvı́ for his help and expertise and reviewers. Any product that may be evaluated in this article, or Dr. Javier Herná ndez (Tumor Tissue Bank, Biobanc HUVH) for claim that may be made by its manufacturer, is not guaranteed the availability of the samples. or endorsed by the publisher. References 1. Varjosalo M, Taipale J. Hedgehog: Functions and mechanisms. Genes Dev 16. Abdel-Salam GMH, Mazen I, Eid M, Ewida N, Shaheen R, Alkuraya FS. 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