# Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development

Khouloud Rjiba, Soumaya Mougou-Zerelli, Imen Hadj Hamida, Ghada Saad, Bochra Khadija, Afef Jelloul, Wafa Slimani, Yosra Hasni, Sarra Dimassi, Hela Ben Khelifa, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Khouloud Rjiba, Soumaya Mougou-Zerelli, Imen Hadj Hamida, Ghada Saad, Bochra Khadija, et al. Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development. Reproductive Biology and Endocrinology, 2023, 21 (1), pp.2. 10.1186/s12958-022-01045-7. hal-04150358

HAL Id: hal-04150358

https://hal.science/hal-04150358

Submitted on 5 Jul 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH

Open Access

<figure><img src="image_3.png" /></figure>

Check for
updates

# Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development

Kholoud Rjiba<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="4" />, Soumaya Mougou-Zerelli<fnref n="1" />,<fnref n="3" />, Imen hadj Hamida<fnref n="1" />, Ghada Saad<fnref n="5" />, Bochra Khadija<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Afef Jelloul<fnref n="1" />, Wafa Slimani<fnref n="1" />,<fnref n="3" />, Yosra Hasni<fnref n="5" />, Sarra Dimassi<fnref n="1" />,<fnref n="3" />, Hela Ben khelifa<fnref n="1" />, Amira Sallem<fnref n="1" />,<fnref n="6" />, Molka Kammoun<fnref n="1" />, Hamza Hadj Abdallah<fnref n="1" />, Moez Gribaa<fnref n="1" />, Joelle Bignon-Topalovic<fnref n="4" />, Sami Chelly<fnref n="7" />, Hédi Khairi<fnref n="8" />, Mohamed Bibi<fnref n="8" />, Maha Kacem<fnref n="5" />, Ali Saad<fnref n="1" />,<fnref n="3" />, Anu Bashamboo<fnref n="4" /> and Kenneth McElreavey<fnref n="4" />*

**Abstract**

**Background** Forty-six, XY Differences/Disorders of Sex Development (DSD) are characterized by a broad phenotypic spectrum ranging from typical female to male with undervirilized external genitalia, or more rarely testicular regression with a typical male phenotype. Despite progress in the genetic diagnosis of DSD, most 46,XY DSD cases remain idiopathic.

**Methods** To determine the genetic causes of 46,XY DSD, we studied 165 patients of Tunisian ancestry, who presented a wide range of DSD phenotypes. Karyotyping, candidate gene sequencing, and whole-exome sequencing (WES) were performed.

**Results** Cytogenetic abnormalities, including a high frequency of sex chromosomal anomalies (85.4%), explained the phenotype in 30.9% (51/165) of the cohort. Sanger sequencing of candidate genes identified a novel pathogenic variant in the *SRY* gene in a patient with 46,XY gonadal dysgenesis. An exome screen of a sub-group of 44 patients with 46,XY DSD revealed pathogenic or likely pathogenic variants in 38.6% (17/44) of patients.

**Conclusion** Rare or novel pathogenic variants were identified in the *AR*, *SRD5A2*, *ZNRF3*, *SOX8*, *SOX9* and *HHAT* genes. Overall our data indicate a genetic diagnosis rate of 41.2% (68/165) in the group of 46,XY DSD.

**Keywords** Disorders of sex development (DSD), 46,XY DSD, Cytogenetic abnormalities, Whole exome sequencing (WES)

<figure><img src="image_4.png" /></figure>

BMC

© The Author(s) 2023. **Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

<footnote>*Correspondence:
Kenneth McElreavey
kenneth.mcelreavey@pasteur.fr</footnote>

<footnote n="1">¹ Laboratory of Human Cytogenetics, Molecular Genetics and Biology of Human Reproduction, Farhat Hached University Teaching Hospital, Sousse, Tunisia</footnote>

<footnote n="2">² Higher Institute of Biotechnology Monastir, University of Monastir, Monastir, Tunisia</footnote>

<footnote n="3">³ Unité de Services Communs en Génétique Humaine, Faculté de Médecine de Sousse, Université de Sousse, Sousse, Tunisia</footnote>

<footnote n="4">⁴ Human Developmental Genetics Unit, CNRS UMR 3738, Institut Pasteur,</footnote>

<footnote>Paris, France</footnote>

<footnote n="5">⁵ Department of Endocrinology, Farhat Hached University Teaching Hospital, Sousse, Tunisia</footnote>

<footnote n="6">⁶ Laboratory of Human Cytogenetics and Biology of Reproduction, Fattouma Bourguiba University Teaching Hospital, Monastir, Tunisia</footnote>

<footnote n="7">⁷ Private Gynecologist Sousse, Sousse, Tunisia</footnote>

<footnote n="8">⁸ Department of Gynecology and Obstetrics, Farhat Hached University Teaching Hospital, Sousse, Tunisia</footnote>
## Background

Differences/Disorders of Sex Development (DSDs) are defined as congenital conditions with a discrepancy between chromosomal, gonadal, and phenotypic sex [1]. They represent a major clinical concern that is most often present in newborns or adolescents [2]. The prevalence of DSD is often underestimated since the diagnosis can be relatively late, at puberty or during adulthood and, in some countries, sexual issues are still sensitive, resulting in a reluctance to seek clinical counselling [3]. This may explain why in Saudi Arabia and Egypt, the incidence of ambiguous genitalia is estimated to be 1:2,500 and 1: 3,000 of live births, respectively, whilst in European countries it is estimated at 1: 4,500–1: 5,500 of live births [4–7]. The data could also reflect the high rate of consanguinity, especially in developing countries, where autosomal recessive forms of DSD are more prevalent [8]. Population isolates may also contribute to the presence of rare or novel variants with a limited geographic range [8].

Forty-six, XY DSD can be due to chromosome abnormalities or genetic variants in the genes involved in the development or function of the male gonad as well as anomalies of downstream target tissues [9]. In most studies, the genetic cause is established in less than 50% of 46,XY DSD cases [1, 9, 10]. At a molecular level pathogenic variants in the *AR*, *NR5A1*, *SRD5A2*, *ZFPM2*, *HSD17B3* and *DHH* genes are the most frequent causes of 46,XY DSD [9, 10]. The aim of this study was to define the genetic etiology in a large cohort of 46,XY DSD patients from a North African population and compare these data to those observed in other populations. The cytogenetic analysis and molecular gene approaches resulted in a combined diagnosis yield of 41.2% (68/165) for this DSD subgroup. Cytogenetic analysis detected autosomal or sex chromosome anomalies in 30.9% of all cases, whereas WES identified rare or novel variants in the *AR*, *SRD5A2*, *ZNRF3*, *SOX8*, *SOX9* and *HHAT* genes (17/44 cases; 38.6%). These results emphasize the usefulness of both cytogenetic approaches as well as exome sequencing to make an accurate genetic diagnosis for a better genetic counseling and knowledge-based management of this group of patients.

## Patients and methods

### Cohort and study design

A total of 165 patients with DSD were referred for genetic consultation in the department of Cytogenetic, Molecular biology, and Biology of Human Reproduction, Teaching hospital Farhat Hached, Sousse, Tunisia over a period

of 3 years (2018–2020). The local Ethics Board of the University Teaching Hospital Farhat Hached approved the present study (IRB00008931) and written consents were taken from adult probands or from the parents when the patient was under 18 years. The patients presented with a range of clinical DSD profiles and their ages ranged from birth to 35 years. They underwent a complete clinical examinations, including genital examination, family history and examination for the presence of somatic abnormalities. Imaging examination and hormonal evaluation were also carried out according to each case. Patients with suspected or confirmed congenital adrenal hyperplasia (CAH) were excluded from this study. All patients are from Tunisian ancestry.

### Genetic analysis

#### Cytogenetic studies

Reverse Heat Giemsa (RHG) banded karyotype was performed on metaphase chromosome preparations obtained from peripheral blood lymphocytes of both patients and parents according to standard protocol (450–550 band level). A minimum of 20 R-banded metaphase chromosomes were analyzed using Cytovision® Karyotyping software version 4.0. Karyotypes were classified according to the International System of Human Cytogenetic Nomenclature (ISCN 2020) [11]. Fluorescent in situ Hybridization (FISH) was carried out on metaphase chromosomes of the patients according to the standard protocol, using commercial probes. Array Comparative genomic hybridization (aCGH) 4 × 44 K micro-arrays was performed using the Agilent platform according to the manufacturer's instructions (Feature Extraction 9.1, CGH Analytics 4.5, Santa Clara, California, United States). An abnormal ratio greater than +0.58 or lower than –0.75 was considered as an alteration. An *in silico* analysis of the unbalanced regions was executed using UCSC Genome Browser (https://genome.ucsc.edu/), the Database of Chromosome Imbalance and Phenotype in Humans using Ensemble Resources (DECI-PHER: https://decipher.sanger.ac.uk/), the Database of Genomic Variants (DGV: http://dgv.tcag.ca/dgv/app/home) and the Online Mendelian Inheritance in Man database (OMIM: https://omim.org/).

#### Sanger sequencing

Genomic DNA was extracted from the peripheral blood of the patient using the FlexiGene DNA Kit (Qiagen, Hilden, Germany). Direct Sanger sequencing was performed using the Big Dye Terminator V3.1 Cycle Sequencing, on the ABI 3730XL sequencer (Applied Biosystems, Foster City, CA, USA). Sequencing data were analyzed by SeqScape 2.0 software (Applied Biosystems).
## Whole exome sequencing

The WES approach was performed on DNA from 44 XY individuals who had a complete clinical investigation including examination of genitalia, hormonal screens and, where possible, gonad histology. All of these patients presented with a broad spectrum of 46,XY DSD phenotypes for which the underlying cause is unknown.

Exonic and adjacent intronic sequences were enriched from genomic DNA using Agilent SureSelect Human All Exon V4, and paired-end sequencing was done with the TruSeq v3 chemistry on Illumina HiSeq2000 platform. Based on the manufacturer's proprietary software, reads were mapped using the Burrows-Wheeler Aligner. Single nucleotide variants (SNV) and small insertions or deletions (Indels) were generated with GATK 1.6 version. BAM files were also carried out using SAMtools version 0.1.18. GATK. Unified Genotyper software was used for calling single nucleotide polymorphism (SNP) and Indels variants for each patient.

The annotated VCF files were then formatted to be used as a Microsoft Excel spreadsheet software and a selection of variants according to well-defined criteria (degree of pathogenicity, type of variant, frequency of the variant in all populations, including sub population) was performed. Synonymous, intronic and non-coding RNA variants were removed. Missense, nonsense, insertion/deletion and splice-site variants that were homozygous with a Minor Allele Frequency (MAF) of > 0.01 were excluded and heterozygous variants with a MAF of > 0.001 according to the GnomAD database (https://gnomad.broadinstitute.org/) were also excluded.

According to the clinical data of each patient, the analysis of variants was performed through a range of web-based bioinformatics tools. The variant Effect Predictor (VEP) bioinformatics tool on the Ensembl website (http://www.ensembl.org/homosapiens/userdata/uploadvariations), gnomAD (https://gnomad.broadinstitute.org/), ClinVar

(https://www.ncbi.nlm.nih.gov/clinvar/) and Database of Genomic Variants (http://dgv.tcag.ca/dgv/app/home) were used to annotate the novel variants.

The possible impact on protein structure and function was evaluated to determine the pathogenicity of the variants based on individual scores made by Sorting Intolerant from Tolerant (SIFT), Polymorphism phenotyping V2 (PolyPhen2) and Rare Exome Variant Ensemble Learner (REVEL; [12]) tools.

The Clustal Omega tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used to generate alignments between three or more protein sequences. The Hope tool was used to analyze the structural effects of a point variation in a protein sequence [13].

Clinical significance was established according to the 2015 American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG) in order to establish a better genotype-phenotype correlation. [14]. Potentially pathogenic variants were verified by Sanger sequencing.

The WES cohort of 46,XY DSD consisted of 19 individuals raised as females and 25 raised as males. Within this group, 17 cases were syndromic and 27 cases non-syndromic cases.

## Results

The most common feature at consultation was atypical external genitalia (67 patients) or typical male external genitalia with azoospermia (42 patients). A total of 30 patients presented with other congenital anomalies including intellectual deficiency, dysmorphic features, heart defects growth delay and cerebral anomalies. Primary amenorrhea and delayed puberty were reported in 18 and 8 cases respectively (Table 1). In this cohort, the patients were classified into three groups: Sex chromosome DSD, autosomal chromosomal abnormalities and 46,XY DSD (Table 1). 66% of the studied patients

<table><caption>**Table 1** Presentation of different categories of studied DSD Tunisian cohort</caption><thead><tr><th>Classification</th><th>Diagnostic criteria</th><th>No</th></tr></thead><tbody><tr><td rowspan="3">Sex chromosome DSD (No = 47)</td><td>Azoospermia</td><td>42</td></tr><tr><td>Atypical genitalia & congenital anomalies</td><td>2</td></tr><tr><td>Atypical genitalia</td><td>3</td></tr><tr><td rowspan="5">Autosomal chromosome anomalies<br>46,XY DSD (No = 109)</td><td>DSD & congenital anomalies</td><td>8</td></tr><tr><td>Atypical genitalia</td><td>64</td></tr><tr><td>Primary amenorrhea</td><td>18</td></tr><tr><td>Delayed puberty</td><td>8</td></tr><tr><td>DSD & congenital anomalies</td><td>20</td></tr></tbody><tfoot><tr><td colspan="3">Total number 165</td></tr></tfoot></table>

Abbreviations: DSD: Disorders of sex development
<figure><img src="image_5.png" /><figcaption>**Fig. 1** Distribution of patients with sex chromosome DSD according to their karyotype. 47,XXY was the most common observation</figcaption></figure>

(110/165) were diagnosed as having 46,XY DSD of whom 23% were raised as females.

## Cytogenetic results

The proportions of different categories of DSD are shown in Table 1 and the distribution of patients with sex chromosome DSD in relation to their karyotype is illustrated in Fig. 1.

Sex chromosome anomalies were detected in 48/165 patients (29.1%) and autosomal chromosome abnormalities were detected in three individuals (1.8%). Klinefelter syndrome (KS) was the most prevalent chromosome sex abnormality in (87.2%) and genetic cause of azoospermia (83.3%) in males.

aCGH was performed on twenty patients based on their clinical presentation, suggesting a contiguous gene syndrome or an a priori assumption of the involvement of rearrangements affecting known gonadal genes or their regulatory sequences with various other extragonadal malformations. In 8 patients, anomalies were observed (Table 2).

These included five cases with intra-chromosomal deletions, two cases of intra-chromosomal duplications, and one case with an inversion duplication/deletion (invdupdel) chromosome imbalance (Table 2). Of these chromosomal anomalies, genes known to cause 46,XY DSD were identified for 4 patients (Table 2) including *DMRT1*, *GATA4* and *NR0B1*. FISH analysis confirmed the heterozygous deletion of the *GATA4* gene in patient 2 and a duplication of the *NR0B1* gene in patient 3. The patient 1 presented the Wolf-Hirschhorn syndrome (WHS) [OMIM#194190]. In addition to the typical WHS phenotype, he presented a hypospadias, micropenis and cryptorchidism. The 4p16.3 deletion presumably results in haploinsufficiency of the *MSX1* gene [OMIM#142983] whose absence might be indirectly responsible for the

hypospadias phenotype as this gene contributes to the spatiotemporal regulation of GnRH transcription during development [15]. In three patients (patients 6–8), there was no obvious candidate gene located within the chromosomal anomaly. Clinical details and cytogenetic results are summarized in Table 2.

## Sequencing data

Exome sequencing was performed in a total of 44 patients with 46,XY DSD. Amongst them, 27 were non-syndromic and 17 presented with somatic anomalies. Of the 44 patients, a genetic cause was established in 17 cases (38.6%) of whom 13 presented non-syndromic DSD form and 4 with syndromic forms. Likely benign (LB) and variants of uncertain significance (VUS) were identified in 11/27 non-syndromic individuals (40.7%) and 4/17 (23.5%) of syndromic individuals. Pathogenic and likely pathogenic variants in the following genes: *AR* (n=6), *SRD5A2* (n=2), *LHCGR* (n=1), *ZNRF3* (n=1), *HHAT* (n=1), *SOX8* (n=1), *IER3IP* 1(n=1), *SRY* (n=1), *SOX9* (n=1), *FLNA* (n=1) and *PEX1* (n=1). The Clinical and molecular findings are summarized in Table 3.

The most common genetic diagnosis was variants in the androgen receptor (26%, 7/27).

A *de novo* pathogenic variant (p.S426*) in the *AR* gene was observed in two sisters who presented complete androgen insensitivity syndrome (CAIS). Two other affected girls with CAIS from unrelated families (DSD3 and DSD4) shared a pathogenic variant (p.G744E), suggesting a possible founder effect. We identified novel or rare likely pathogenic variants in the *ZNRF3* (DSD 11), *HHAT* (DSD 12), and *SOX8* genes (DSD 30). A girl with 46,XY complete gonadal dysgenesis carried novel mis-sense heterozygous *ZNRF3* variant (p.I338M). According to SIFT (0.01), PP2 (0.519) and REVEL (0.461) scores, this variant is likely to be disease causing. Isoleucine 338 is a highly conserved residue within the long
<table><caption>**Table 2** Clinical and cytogenetic findings in patients with DSD explored with aCGH (4 × 44 K)</caption><thead><tr><th>Patent</th><th>Patient<sup>1</sup></th><th>Patient 2</th><th>Patient 3</th><th>Patient 4</th><th>Patient 5</th><th>Patient 6</th><th>Patient 7</th><th>Patient 8</th></tr></thead><tbody><tr><td>Indication</td><td>Dysmorphic features</td><td>24 years</td><td>7 months</td><td>8 months and half</td><td>3 years</td><td>9 months</td><td>3 Years</td><td>1 year</td></tr><tr><td>Consanguinity</td><td>No</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>Abnormal external genitalia</td><td>Abnormal external genitalia</td><td>No</td></tr><tr><td>Sex of rearing</td><td>M</td><td>F</td><td>F</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td></tr><tr><td>congenital anomalies associated</td><td>Dysmorphic features<br>Microcephaly<br>Inter-atrial communication heat defect</td><td>Mental retardation</td><td>Hypotonia, Dysmorphic features, psychomotor delay<br>Inter-atrial communication heat defect</td><td>Dysmorphic features<br>Microcephaly<br>Psychomotor delay</td><td>Dysmorphic features<br>Microcephaly</td><td>Dysmorphic features<br>microcephaly</td><td>Dysmorphic features<br>microcephaly</td><td>Dysmorphic features<br>Inter-atrial communication heat defect</td></tr><tr><td>Genitalia</td><td>External<br>Internal</td><td>Micropenis, hypospadias, cryptorchidism</td><td>Female external genitalia</td><td>micropenis</td><td>micropenis</td><td>Microcephaly</td><td>Microcephaly</td><td>Arterial hypertension</td></tr><tr><td>Gonads</td><td>Testis</td><td>Micropenis, palpable right testis</td><td>Absence of ovaries and uterus</td><td>NA</td><td>NA</td><td>NA</td><td>Microcephaly</td><td>Microcephaly</td></tr><tr><td>Karyotype</td><td>46,XY(der4)t(16,3)</td><td>46,XY</td><td>Absence of ovaries and uterus</td><td>Testis</td><td>Testis in inguinal position</td><td>Testis in the abdomen</td><td>Testis in inguinal position</td><td>Small testis</td></tr><tr><td>FISH (SRY gene)</td><td>46,XY(der4)t(16,3)<br>(SRY × 1)</td><td>46,XY,ish(Yp11.3)(SRY × 1)</td><td>46,XY,der(Y)(ish(Yp11.3)(SRY × 1)</td><td>46,XY,der(Y)(9)<br>(SRY × 1)</td><td>46,XY,ish(Yp11.3)(SRY × 1)</td><td>45,XY,rob(13.1)(q10q10)</td><td>46,XY,der(t12)<br>(4/46,XY,t(16))</td><td>46,XY,ish(Yp11.3)(SRY × 1)</td></tr><tr><td>aCGH (4 × 44 K)</td><td>46,XY,arf(NCB36)<br>Hp.63.62(447_9,065,571) × 1 dn<br>(deletion size: 4.8 Mb)</td><td>46,XY,arf8p231(NCB36)<br>(7,290,597_11,665,267) × 1<br>(deletion size: 4 Mb)</td><td>46,XY,der(Y)(Yp21.3p11.3)<br>NCB36xp21.20(2,710,316_30,233,793)<br>X2 dn<br>(duplication size: 27.5 Mb)</td><td>46,XY,der(Y)(9)<br>(deletion size: 1 Mb)</td><td>46,XY,arf(NCB36<br>9p22.2)(601_628_7,786,728)<br>(deletion size ~ 6 Mb)</td><td>45,XY,rob(13.14)arf(NCB36<br>8p23.20(18461_7,290,647) × 1,<br>8p21.20(16,267,630<br>-30,89917) × 3<br>deletion size ~ 7 Mb<br>(duplication<br>duplication size ~ 18 Mb)</td><td>46,XY,der(t12)<br>(4/46,XY,der(t12)<br>(4/46,XY,t(16))ish(Yp11.3)<br>(SRY × 1)</td><td>46,XY,arf(NCB36<br>16p12.12)(29,582,349_30,106,011) × 1<br>(Deletion size: 1 Mb)</td></tr><tr><td>FISH Specific loci Probes</td><td>46,XY(der4)t(16,3)<br>(WHSC1 × 1)</td><td>Blue FISH(RP11-52819)<br>46,XY,ish(8p23) (RP11-52819 × 1)</td><td>46,XY,der(Y)(Xp21.2)(NR0B1 × 2)<br>46,XY,der(Y)(ish(MCP22_3)KAI1 × 2)<br>46,XY,der(Y)(ish(WCPX × 1)WCPY × 1)</td><td>46,XY,ish(8p23)<br>(subre18q × 2)</td><td>46,XY,ish(8p23)<br>(subre18q × 2)</td><td>NA</td><td>46,XY,ish(8p23)<br>(subre18q × 2)</td><td>NA</td></tr><tr><td>Inheritance</td><td>De novo</td><td>NA</td><td>De novo</td><td>NA</td><td>De novo</td><td>De novo</td><td>De novo</td><td>NA</td></tr><tr><td>Initial clinical diagnosis</td><td>Wolf Hirshhorn Syndrome</td><td>46,XY, GD</td><td>46,XY, GD</td><td>Alf syndrome</td><td>Alf syndrome</td><td>46,XY, DSD</td><td>De novo</td><td>NA</td></tr><tr><td>Candidate gene in association with the DSD phenotype</td><td>MSX1 (candidate gene)</td><td>GATA4</td><td>NR0B1</td><td>DMRT1-3</td><td>DMRT1-3</td><td>NA</td><td>NA</td><td>NA</td></tr></tbody></table>

Abbreviations: NA: not available; M: male, F: female; dn: de novo, DSD: disorders of sex development; FISH: Fluorescence in situ hybridization; aCGH: array comparative genomic hybridization
<table><caption>**Table 3** Clinical phenotype, hormonal profil and details of potentially pathogenic variants identified by WES in a cohort of individuals with 46,XY DSD</caption><thead><tr><th>nDSD case</th><th>Age/ sex of rearing</th><th>FSH (UI/N)/ range</th><th>LH (UI/N)/ range</th><th>Testosterone (ng/ml)/ Normal range</th><th>Genitourinary External genitalia</th><th>Internal genitalia</th><th>Gonad position</th><th>Gonad Histology</th><th>Somatic features</th><th>Gene/Variant/Zygosity/ inheritance</th><th>MAF and population (gnomAD)/ Predicted effect on protein</th><th>Clinical significance: ACMG/ClinVar/ GV, Ref</th></tr></thead><tbody><tr><td>DSD1</td><td>17Y/F</td><td>8.92 (6.3-24)</td><td>8.81 (9.6-80)</td><td>4.06 (0.7-19)</td><td>Micropenis, absence of labia minora, no vagina</td><td>Absent MD, presence of seminal vesicles, hypotrophic testis</td><td>R: inguinal</td><td>Testis/ NA</td><td>-</td><td>SRD5A2:NM_000348c. G344Ap.G15D:Hom, parents</td><td>0.001/1964: Latino-Admixed-American/ SIFT: D (0.03), PP2: PD (0.993), REVEL: NA</td><td>P/NA/ P [Lavinia vija et al 2014, Laurent Maimoun 2011]</td></tr><tr><td>DSD2</td><td>28Y/F</td><td>NA</td><td>NA</td><td>NA</td><td>Micropenis, absence of labia minora and majora, vaginal aplasia</td><td>Absent MD, atrophic testes</td><td>R: inguinal</td><td>Testis/ Sertoli cells, no germ cells, Leydig cell hyperplasia</td><td>-</td><td>SRD5A2:NM_000348c. A622C:PT208P, Hom, parents</td><td>Novel/SIFT: SIFT: D(0.01), PP2: PD (0.913), REVEL: NA</td><td>LP/NA/ NA</td></tr><tr><td>DSD3</td><td>30Y/F</td><td>7 (1.7-12)</td><td>15.5 (1.1-7)</td><td>NA</td><td>F</td><td>Absent uterus, hypoplastic gonads</td><td>NA</td><td>NA</td><td>-</td><td>ARNM_000044c.G2231Ap. G744E, Hemi, maternal</td><td>NA/ SIFT: D(0.00), PP2: PD(1.00), REVEL: 0.935 (LDC)</td><td>P/ P/ NA</td></tr><tr><td>DSD4</td><td>8Mo/F</td><td>NA</td><td>0.8 (2-12)</td><td>&lt;0.1 (0.2-0.8)</td><td>F, bilateral inguinal hernia</td><td>Absent MD, presence of testes in hernia sac</td><td>Hernia sac</td><td>Testis/ Sertoli cells with seminiferous tubes, no germ cells</td><td>-</td><td>ARNM_000044c.G2231Ap. G744E, Hemi, NA</td><td>NA/ SIFT: D(0.00), PP2: PD(1.00), REVEL: 0.935 (LDC)</td><td>P/ P/ NA</td></tr><tr><td>DSD5</td><td>4D/F</td><td>NA</td><td>NA</td><td>NA</td><td>Hypertrophy of the clitoris and labia majora</td><td>NA</td><td>NA</td><td>NA</td><td>-</td><td>ARNM_000044c.A1745Gp. KS81R, Hemi, maternal</td><td>Novel/ SIFT: D (0.00), PP2: PD (0.998), REVEL: NA</td><td>LP/NA/ NA</td></tr><tr><td>DSD6</td><td>18Y/F</td><td>NA</td><td>NA</td><td>NA</td><td>F</td><td>Absent MD, presence of bilateral gonadal mass with epididymes</td><td>NA</td><td>Two atrophic testes, presence of Sertoli cells, no germ cells</td><td>-</td><td>ARNM_000044c.G159T/p. G533*, Hemi, maternal</td><td>Novel/LOF</td><td>P/ NA/NA</td></tr><tr><td>DSD7<br>DSD8<br>(sibs)</td><td>25Y/F</td><td>28.7 (1.1-7)</td><td>5.6 (1.1-7)</td><td>NA</td><td>F, bilateral inguinal hernia</td><td>Vagina present, absent MD</td><td>NA</td><td>no residual gonad</td><td>-</td><td>ARNM_000044c.C1277A:p. S426*, Hemi, De novo</td><td>Novel/LOF</td><td>P/ NA/ NA</td></tr><tr><td>DSD9</td><td>1Y and half/M</td><td>NA</td><td>NA</td><td>NA</td><td>Hypospadias, cryptorchidism, incomplete foreskin</td><td>NA</td><td>NA</td><td>NA</td><td>-</td><td>ARNM_000044c.T170A:p. L57Q, Hemi, maternal</td><td>Novel/ SIFT: D (0.001), PP2: unknown, REVEL: 0.150(B)</td><td>B/LB/NA</td></tr><tr><td>DSD10</td><td>14Y</td><td>39(1.7-12)</td><td>NA</td><td>NA</td><td>F</td><td>Absent gonads and uterus</td><td>NA</td><td>No residual gonad</td><td>-</td><td>LHCGRNNM_000233c. C1573T:Q525*, Hom, parents</td><td>NA/LOF</td><td>P/NA/ P [Imen et al 2015]</td></tr><tr><td>DSD11</td><td>19Y/F</td><td>25(6.3-24)</td><td>44(29.6-60)</td><td>NA</td><td>F</td><td>Absent uterus, vagina present, R ovary: 18 mm/L ovary:26 mm</td><td>NA</td><td>Ovarian-like tissue</td><td>-</td><td>ZNRF3:NM_001206998c. A1014Gp.I38M, Het, NA</td><td>Novel/ SIFT: D (0.01)/ PP2:PD(0.519)/ REVEL: 0.461(LB)</td><td>LP/NA/NA</td></tr></tbody></table>
<table><caption>Table 3 (continued)</caption><thead><tr><th>nDSD case</th><th>Age/ sex of rearing</th><th>FSH (UI/I)/ Normal range</th><th>LH (UI/I)/ Normal range</th><th>Testosterone (ng/ml)/ Normal range</th><th>Genitourinary External genitalia</th><th>Internal genitalia</th><th>Gonad position</th><th>Gonad Histology</th><th>Somatic features</th><th>Gene/Variant/Zygosity/ inheritance</th><th>MAF and population (gnomAD)/ Predicted effect on protein</th><th>Clinical significance: ACMG/ ClinVar/ GV, Ref</th></tr></thead><tbody><tr><td>DSD12</td><td>40D/F</td><td>NA</td><td>NA</td><td>N/A</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>Hydrocephalus, skeletal malformations, bilateral anophthalmos, agenesis of the corpus callosum</td><td>HHATNM_018194.6c. C934Ap.sp.R312S, Horn, parents</td><td>0.00005482 European/ SIFT(0.06)/ PP2: PD(0.459)/ REVEL:0.159(B)</td><td>LP/N/A/NA</td></tr><tr><td>DSD13</td><td>3 Mo/M</td><td>NA</td><td>NA</td><td>NA</td><td>Micropenis, hypospadias, cryorchidism</td><td>NA</td><td>NA</td><td>NA</td><td>Dysmorphic features, diabetes, epileptic seizures, atrophy of the supra tentorial level</td><td>IER3P1: NM_016097.5c. T62Gp.V21G/Horn, AR parents</td><td>0.0001491 Latino admixed American /SIFT(0.00)/ PP2:PD(0.583)/ REVEL:0.886(LDC)</td><td>P/VUS/Khouloud et al. 2021</td></tr><tr><td>DSD14</td><td>23Y/F</td><td>103.53(17-95)</td><td>15.99(8-33)</td><td>NA</td><td>F</td><td>No ovaries, small uterus</td><td>No residual gonad</td><td>NA</td><td>NO</td><td>NSMFexon3c.134-4C&gt;T, Het, NA</td><td>Novel/LOF</td><td>LB/N/A/NA</td></tr><tr><td>DSD15</td><td>1Y/M</td><td>NA</td><td>NA</td><td>NA</td><td>Micropenis, hypospadias, small testes</td><td>NA</td><td>NA</td><td>NA</td><td>Dysmorphic features, psychomotor delay, hypotrophy</td><td>PORNNM_000941c.G1736Ap. R579Q, Het, NA</td><td>European 0.0001176/ SIFT(0.16)/ PP2:R(0.121)/ REVEL:0.187(B)</td><td>B/NA/NA</td></tr><tr><td>DSD16</td><td>16Y/F</td><td>48.33(6.3-24)</td><td>20.20(29.6-60)</td><td>NA</td><td>F</td><td>Infantile uterus, no ovaries</td><td>No residual gonad</td><td>NA</td><td>-</td><td>KDM3A/NM_00114688c. C1535Tp.S512L, Het, NA</td><td>SIFT(0.13)/ PP2:R(0.2)/ REVEL:0.371(B)</td><td>LB/N/A/NA</td></tr><tr><td>DSD17</td><td>2Y/M</td><td>NA</td><td>NA</td><td>NA</td><td>Left testicular ectopy</td><td>NA</td><td>NA</td><td>NA</td><td>Dysmorphic features/partial agenesis of the corpus callosum/microcephaly</td><td>PEX1NM_000466c.G2528Ap. G843D, Horn, parents</td><td>0.00001470/ SIFT(0.05)/ PP2:R(0.009)/ REVEL:0.426(LB)</td><td>LP/N/A/NA</td></tr><tr><td>DSD18</td><td>1Y and 1Mo/M</td><td>NA</td><td>NA</td><td>NA</td><td>Hypospadias, micropenis, unilateral cryptorchidism</td><td>NA</td><td>R in place Li in the abdomen</td><td>Testes/NA</td><td>Dysmorphic features, CN/intra-uterine growth delay</td><td>POLENM_006231c.C1707Gp. F569LHet, parents</td><td>African American 0.00009653/ SIFT(0.0)/PP2: PD(1.0)/REVEL:0.984(LDC)</td><td>P/P/N/A</td></tr><tr><td>DSD19</td><td>4Mo/M</td><td>NA</td><td>NA</td><td>NA</td><td>Micropenis, cryptorchidism, small smooth scrotum</td><td>NA</td><td>NA</td><td>-</td><td>ANOS1:NM_000216c. C1283Tp.P428L, Hemi, parents</td><td>European 0.00005634/ SIFT(0.73)/PP2:R(0.00)/ REVEL:0.081(B)</td><td>LB/LB/NA</td></tr></tbody></table>
<table><caption>Table 3 (continued)</caption><thead><tr><th>nDSD case</th><th>Age/ sex of rearing range</th><th>FSH (UI/N)/ Normal range</th><th>LH (UI/II)/ Normal range</th><th>Testosterone (ng/ml)/ Normal range</th><th>Genitourinary External genitalia</th><th>Internal genitalia</th><th>Gonad position</th><th>Gonad Histology</th><th>Somatic features</th><th>Gene/Variant/Zygosity/ inheritance</th><th>MAF and population (gnomAD)/ Predicted effect on protein</th><th>Clinical significance: ACMG/ClinVar/ GV, Ref</th></tr></thead><tbody><tr><td>DSD20</td><td>15D/M</td><td>NA</td><td>NA</td><td>N/A</td><td>Anterior interscrotal hypoplasias, micropenis, hypoplastic scrotum, left testicular ectopy</td><td>Bilateral hydrocele</td><td>R: no residual gonad, Lingual region</td><td>Testis/NA</td><td>-</td><td>FGF17/NM_001304478c. C32T/p.T11I, Het, NA</td><td>0.00001470/euro-pan/ SIFT(0.17)/ PP2B(0.053)/ REVEL:0.256(B)</td><td>VUS/NA/NA</td></tr><tr><td>DSD21</td><td>36Y/M</td><td>116(1.5-10)</td><td>NA</td><td>0.52(2.5-10)</td><td>Small left testis</td><td>R: no testis L: atrophic testis</td><td>Testis/NA</td><td>-</td><td>NSMF/NM_015537/c.A125Gp. N42S, Het, NA</td><td>Novel/ SIFT(0.005)/ PP2B(0.417)/REVEL: 0.069(B)</td><td>B/NA/NA</td></tr><tr><td>DSD22</td><td>4Y and 9M2/M</td><td>NA</td><td>NA</td><td>N/A</td><td>Micropenis, small scrotum</td><td>Testis in the inguinal region</td><td>Ringuinal/Lingual</td><td>Testis/NA</td><td>Epilepsy;coortical atrophy</td><td>PDYN/NM_024411c.34delCp: L12S, Het, NA</td><td>Novel</td><td>VUS/NA/NA</td></tr><tr><td>DSD23</td><td>21Y/M</td><td>NA</td><td>NA</td><td>N/A</td><td>Hypospadias, small testis</td><td>Testis in scrotum</td><td>NA</td><td>Testis</td><td>Hypothyroidism/low hairness</td><td>MAMLDT1/NM_001177465c. C2573T/p.P858L, Het, maternal</td><td>European 0.0001506/SIFTD/ PP2:PD/REVEL: 0.188(B)</td><td>VUS/VUS/NA</td></tr><tr><td>DSD24</td><td>4Y and 6M2/M</td><td>NA</td><td>NA</td><td>N/A</td><td>Hypospadias, cryptorchidism</td><td>NA</td><td>L & R: abdomen</td><td>Testis/NA</td><td>-</td><td>ARID1B/NM_001346813 c.1053_1054insGGCp. G351delinsCG Het, NA</td><td>Novel/LOF</td><td>VUS/VUS/NA</td></tr><tr><td>DSD25</td><td>15Y/F</td><td>NA</td><td>NA</td><td>N/A</td><td>F</td><td>Immature uterus without uterine cavity, vagina</td><td>No residual gonad</td><td>NA</td><td>-</td><td>, NSRNM_000208c.C3034Ap. V1012M, Het, NA</td><td>European 0.00008790/SIFTD/ PP2:PD/REVELNA East Asian 0.0001930 SIFTD(0.02)/ PP2:PD(0.546)/REVEL: 0.634(LDC)</td><td>VUS/VUS/NA</td></tr><tr><td>DSD26</td><td>1Y and half/M</td><td>NA</td><td>NA</td><td>N/A</td><td>Micropenis, cryptorchidism</td><td>NA</td><td>NA</td><td>NA</td><td>Dysmorphic features</td><td>NBN/NM_002485.5c.C798T: p.R215W, Het, NA</td><td>African/American 0.00008078/ SIFTD(0.01)/ PP2:PD(0.366)/ REVEL:0.538(LDC)</td><td>LP/VUS/NA</td></tr><tr><td>DSD27</td><td>39Y/M</td><td>NA</td><td>NA</td><td>N/A</td><td>Micropenis</td><td>No testes</td><td>NA</td><td>NA</td><td>-</td><td>AMH-NM_000479c.C553Gp. Q185E, Het, NA</td><td>European 0.001985/ SIFTD(0.00)/ PP2:PD(0.977)/ REVEL: 0.343(B)</td><td>LB/VUS/NA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>POR/NM_000941c.C344Tp. A115V, Het, NA</td><td>European 0.0001911/ SIFT(0.041)/ PP2B(0.408)/ REVEL:0.481(LB)</td><td>LB/LB/NA</td></tr></tbody></table>
<table><caption>**Table 3 (continued)**</caption><thead><tr><th>nDSD case</th><th>Age/ sex of rearing</th><th>FSH (UI/I)/ Normal range</th><th>LH (UI/I)/ Normal range</th><th>Testosterone (ng/ml)/ Normal range</th><th>Genitourinary External genitalia</th><th>Internal genitalia</th><th>Gonad position</th><th>Gonad Histology</th><th>Somatic features</th><th>Gene/Variant/Zygosity/ inheritance</th><th>MAF and population (gnomAD)/ Predicted effect on protein</th><th>Clinical significance: ACMG/ClinVar/ GV, Ref</th></tr></thead><tbody><tr><td>DSD28</td><td>1Y and half/M</td><td>NA</td><td>NA</td><td>NA</td><td>Unilateral cryorchidism</td><td>NA</td><td>Rabdomen L: scrotum</td><td>NA</td><td>Dysmorphic features: mental retardation, clinodactyly, patents</td><td>FRAS1_NM_025074c. A7622Gp2_N25415; Horn, patents</td><td>European 0.0035442/ SIFT: D(0.03)/polyphen2: PD(0.997) / REVEL:0.177(B)</td><td>B/LB/NA</td></tr><tr><td>DSD29</td><td>8Mo/M</td><td>NA</td><td>NA</td><td>NA</td><td>Hypospadias/cryptorchidism</td><td>Testes in inguinal region</td><td>L & R inguinal region</td><td>Testes/NA</td><td>Dysmorphic features: hypotonia/microcephaly/fallot tetralogy/brachycaphaly</td><td>ANKRD11_NM_001256183c. C55781p.P18605, Het, NA</td><td>European 0.0005586/ SIFT: T(0.07)/ PP2: LB(0.348)/ REVEL:0.148(B)</td><td>LB/LB/NA</td></tr><tr><td>DSD30</td><td>21Y/F</td><td>32.6(3-15)</td><td>53.3(1.2-12.5)</td><td>NA</td><td>F</td><td>Absent vagina and uterus</td><td>No residual gonad</td><td>NA</td><td>-</td><td>SOX8_NM_014587c.A676Cp. T226P, Het, NA</td><td>Novel/ SIFT(D(0.04)/ PP2: PD(0.99)/ REVEL:0.812(LDC)</td><td>LP/NA/NA</td></tr><tr><td>DSD31†</td><td>33Y/F</td><td>57(3-12)</td><td>11.7(11-7)</td><td>0.4(3-12)</td><td>F</td><td>Bilateral gonadal agenesis</td><td>No residual gonad</td><td>Small impubertal uterus, Fallopian tubes</td><td>-</td><td>SRYNM_003140.3c.C188Ap. P63H, Hemi, NA</td><td>Novel/ SIFT(D(0.00)/ PP2: PD(1.00)/ REVEL:0.720(LDC)</td><td>LP/NA/NA</td></tr></tbody></table>

Abbreviations: **ACMG** american college of medical genetics, **AD** autosomal dominant, **AMH** anti-Müllerian hormone, **AR** autosomal recessive, **B** benign, **D** day, **D** deleterious, **DSD** disorders/differences of sex development, **F** female, **FSH** follicle stimulating hormone, **gnomAD** genome aggregation database, **GV** gene variants previously associated with the disease, **Hemi** hemizygous, **Het** heterozygous, **Hom** homozygous, **L** left, **LB** likely benign, **LDC** likely disease causing, **LH** luteinizing hormone, **LOF** loss-of-function, **LP** likely pathogenic, **LPG** left palpable gonad, **M** male, **MAF** minor allele frequency, **MD** Mullerian ducts, **Mo** month, **NA** not available, **P** pathogenic, **PD** probably damaging, **R** right, **Ref** reference, **REVEL** rare exome variant ensemble learner, **Sibs** siblings, **VUS** variant of uncertain significance, **WES** Whole exome sequencing, **Y** year, * identified with direct sanger sequencing
<figure><img src="image_6.png" /><figcaption>**Fig. 2** A Schematic representation of ZNRF3 protein indicating the known functional domains. The sequence alignment indicating the position and evolutionary conservation of the mutated isoleucine 338 residue, immediately adjacent to the RING finger domain. Previously published variants linked to 46,XY DSD are shown and located within the intracellular domain. B Schematic representation of HHAT protein indicating the position of the mutated p.R312 residue. Other published variants associated with this syndromic form of 46,XY DSD are indicated. C Representation of the SOX8 protein showing the position of the mutated p.T226 residue located in the evolutionary conserved TA1 domain. The only other SOX8 variant known to be associated with 46,XY DSD is the p.E156D mutation located within the HMG-box. Right, the mutated threonine residue is conserved in the SOXE group of proteins. DIM, DNA-dependent dimerization domain; HMG, high mobility group; MBOAT, Membrane Bound O-Acyltransferase domain; TA, transactivation domain; TM, transmembrane domain; SP, signal peptide</figcaption></figure>

intracellular domain (Fig. 2A), immediately adjacent to the ring domain (amino acids 293–334), which is responsible for the E3 ubiquitin ligase activity. A newborn 46,XY girl (DSD 12) presented hydrocephalus, skeletal malformations, bilateral anophtalmos and agenesis of the corpus callosum carried a very rare homozygous variant (p.R312S) in *HHAT* gene (DSD 12). The evolutionary conserved p.R312 residue is located in the Membrane Bound O-Acyltransferase domain 2 (MBOAT 2; Fig. 2B), which is required to palmitoylate Hedgehog proteins including SHH and DHH [16]. The *in silico* tools PP2 (0.99), SIFT (0.04) and REVEL (0.812) showed that this variant is likely to be disease causing. Hope tool predicted this variant to be damaging for the protein since the mutation introduces a more hydrophobic residue at this position and this can result in loss of hydrogen bonds and/or disturb correct folding. A novel heterozygotic p.T226P variant in *SOX8* gene was identified in a 46,XY female with probable testicular regression syndrome (high FSH, LH levels, no residual gonad, absent vagina and uterus). The T226 residue, located within the transactivation domain 1, is highly conserved among vertebrates and within the

SOXE group of proteins (Fig. 2C). PP2 prediction tool indicated that this variant is likely disease causing. Hope predicted this variant to be likely damaging to the protein since it is located in an important domain for the main activity of the protein. The charge of the wild-type residue will be lost, and that change can cause loss of interactions with other molecules or residues. The inheritance pattern of both the *ZNRF3* and *SOX8* variants is unknown as parents DNA was unavailable. Both variants are absent from all public databases.

A rare homozygous variant in *PEX1* gene (p.G843D) was identified in a child boy (DSD 17) with syndromic form of DSD, including microcephaly, partial agenesis of the corpus callosum, dysmorphic features and unilateral cryptorchidism. SIFT (0), PP2 (1) and REVEL (0.984) prediction tools indicated that this variant is likely disease causing.

## Discussion

Sex chromosome as well as autosomal anomalies were present in 30.9% of the 46,XY DSD cohort, with the majority classified as 47,XXY Klinefelter's syndrome. This
is similar to frequencies reported by Mazen et al., 2021 studying a North African cohort, but higher than those reported in other studies [17]. As suggested by Mazen et al., 2021, this rate may be due to a recruitment bias as the research center in Tunisia is a reference centre for cytogenetics. However, it indicates that a considerable proportion of 46,XY DSD cases is due to chromosomal anomalies that can be detected during routine karyotyping. aCGH detected further 8 individuals with chromosomal anomalies, associated with 46,XY DSD in 4 patients.

WES is considered the best method for identifying disease causing gene variants in DSD due to the complexity of the phenotypes [18]. Current data indicate that approximately more than half of patients with 46,XY DSD still lack a definite clinical diagnosis at the genetic level after WES [10, 19]. In this North African cohort of DSD, the genetic cause was established in 41.2% (68/165) of the total cohort, with a genetic cause identified in 38.6% of patients following WES. Recent cohort studies, using WES rather than targeted NGS panels have given a diagnosis yield in 46,XY DSD cohorts of 43% and 51% respectively [10, 20]. The lower yield of 38.6% reported here may reflect the proportion of undervirilised men in the cohort, a group that is difficult to reach a definitive clinical diagnosis or establish a genetic etiology [21, 22]. However, similarly to other studies the most common genetic cause was hemizygous variants in the AR [23, 24]. A total of 7 individuals, including two sisters, carried pathogenic variants in the AR. The G744E variant was observed in two unrelated patients, suggesting a possible founder effect for this variant.

A proportion of XY males carrying deletions of 8p23.1 that encompasses the *GATA4* gene have hypospadias and bilateral cryptorchidism [25, 26]. Here, a 46,XY female with atypical external genitalia (micropenis, small palpable right testis) carried a 4 Mb microdeletion in the 8p23.1 encompassing the *GATA4* gene [27, 28]. Pathogenic variants in *GATA4* have been identified in 46,XY DSD with or without cardiac heart defect [27–29]. To our knowledge this is the first case with a 8p23 microdeletion in a patient with 46,XY DSD raised as female.

WES revealed several very rare causes of 46,XY DSD including the genes *ZNRF3*, *SOX8* and *HHAT*. A novel heterozygous missense variant (p.I338M) in *ZNRF3* was identified in a 46,XY female with complete gonadal dysgenesis (DSD11). *ZNRF3* functions in testis-determination by inhibiting canonical pro-ovary WNT signaling pathway in XY gonads [30]. *ZNRF3* does this by targeting Frizzled receptors for degradation by ubiquitination and increased membrane turnover [31]. A total of four rare or novel heterozygous variants (3 missense and one splice

region) in *ZNRF3* have been reported with both mild and severe 46,XY DSD [30]. All of these variants, including the p.I338M reported here, are located within the C-terminal intracellular domain portion of the protein [31], suggesting a possible genotype/phenotype correlation. *SOX8* is an high mobility group (HMG)-box transcription factor, which is co-expressed with *SOX9* and *NR5A1/SF1* in testis-determination. *SOX8* shows functional redundancy with *SOX9* and may represses Foxl2 expression [32–34]. Heterozygous missense variants in *SOX8* are associated with either male or female infertility. Although rearrangements at the *SOX8* locus are associated with 46,XY gonadal dysgenesis, only a single pathogenic missense variant, located within the conserved HMG domain (p.E156D), has been demonstrated to cause 46,XY gonadal dysgenesis [35]. Here, a novel heterozygous missense variant p.T226P, located within transactivation (TA) domain, was carried by a 46,XY female with testicular regression syndrome. The p.T226 residue is conserved within the SOXE group of proteins, suggesting a functional role. The mode of inheritance of the *ZNRF3* and *SOX8* variants mutation is unknown, as the parents were unavailable for study. Hedgehog acyltransferase (HHAT) is an ER-resident multipass membrane protein consisting of 10 transmembrane domains and 2 re-entrant loops [36]. It is a member of the membrane bound-O-acyltransferase (MBOAT) family of enzymes that catalyze the attachment of specific fatty acids to secreted proteins [37]. *Hhat*⁻/⁺ mice display severely impaired development of fetal Leydig cells, Sertoli cells and testis cords[16]. In humans, biallelic pathogenic variants in *HHAT* are very rare and associated with a wide spectrum of neurodevelopmental phenotype including microcephaly, cerebellar vermis hypoplasia, gonadal dysgenesis, seizures and thinning of corpus callosum [16, 38, 39]. Only four families have been described in the literature and the common features are microcephaly and gonadal dysgenesis. Here, we identified a novel homozygous missense variant (p.R312S) in the conserved MBOAT domain-2 of *HHAT* carried by a 46,XY female with somatic anomalies including hydrocephalus, agenesis of the corpus callosum, skeletal malformations and bilateral anophtalmia.

## Conclusion

A combination of cytogenetics and exome sequencing can explain the genetic cause of 46,XY DSD in just over 40% of all cases. Exome sequencing is particularly useful in detecting very rare genetic causes of DSD in genes such as *ZNRF3*, *SOX8* or *HHAT* that would otherwise have been difficult to determine using other approaches.
**Abbreviations**

aCGH Array comparative genomic hybridization

DNA Deoxyribonucleic Acid

DSD Differences/disorders of sex development

HMG High mobility group

KS Klinefelter syndrome

WES Whole exome sequencing

**Acknowledgements**

The authors would like to express their sincere gratitude to all patients and their parents for their participation in this work. We are grateful to the associated clinicians for providing detailed clinical information and we also thank Mhadheb Msaoura for English editing.

**Authors' contributions**

K.R carried out the cytogenetic and molecular genetic studies, analyzed the datasets, and performed protein sequence alignment. I.H, W.S, H.B.K and J.B.T participated in some experiments. G.S, A.J, Y.H, S.D, A.S, M.K, H.A, M.G, H.K, M.B, S.C, M.K and A.S provided detailed clinical data and critically evaluated the manuscript. S.M.Z, A.B and K.M. designed the study, drafted the final manuscript, and analyzed datasets.

**Funding**

This work is funded in part by a research grant from the European Society of Pediatric Endocrinology, a scholarship from the Tunisian government, and by the Agence Nationale de la Recherche (ANR), ANR-10-LABX-73 REVIVE, ANR-17-CE14-0038-01, ANR-19-CE14-0022, ANR-20-CE14-0007 and ANR-19-CE14-0012.

**Availability of data and materials**

Please contact the author for data requests.

**Declarations**

**Ethics approval and consent to participate**

The local Ethics Board of the University Teaching Hospital Farhat Hached approved the present study (IRB00008931) and written consents were taken from the parents for data publication.

**Consent for publication**

Consent for publication of all patients has been obtained.

**Competing interests**

The authors declare that there are no competing financial interests in relation to the work described.

Received: 14 July 2022 Accepted: 1 December 2022

Published online: 11 January 2023

**References**

1. Hughes IA. Disorders of sex development : a new definition and classification. Best Pract Res Clin Endocrinol Metab. 2008;22(1):119-34.

2. Ostrer H. Disorders of sex development (DSDs): An update. J Clin Endocrinol Metab. 2014;99(5):1503-9.

3. Acién P, Acién M. Disorders of sex development: Classification, review, and impact on fertility. J Clin Med. 2020;9:1-33.

4. Abdullah MA, Katugampola M, Al-Habib S, Al-Jurayyan N, Al-Samarrai Al-Nuaim AA, Patel PJ, et al. Ambiguous genitalia: Medical, socio-cultural and religious factors affecting management in Saudi Arabia. Ann Trop Paediatr. 1991;11(4):343-8.

5. Mazen I, Hiort O, Bassiouny R, El Gammal M. Differential diagnosis of disorders of sex development in Egypt. Horm Res. 2008;70(2):118-23.

6. Thyen U, Lanz K, Holterhus PM, Hiort O. Epidemiology and initial management of ambiguous genitalia at birth in Germany. Horm Res. 2006;66(4):195-203.

7. Sax L. How common is intersex? A response to Anne Fausto-Sterling. J Sex Res. 2002;39(3):174-8.

8. Bashamboo A, McElreavey K. Consanguinity and disorders of sex development. Hum Hered. 2014;77(1-4):108-17.

9. Elzaia M, McElreavey K, Bashamboo A. Genetics of 46, XY gonadal dysgenesis. Best Pract Res Clin Endocrinol Metab. 2022;36(1):101-633.

10. Globa E, Zelinska N, Shcherbak Y, Bignon-Topalovic J, Bashamboo A, McElreavey K. Disorders of Sex Development in a Large Ukrainian Cohort: Clinical Diversity and Genetic Findings. Front Endocrinol (Lausanne). 2022;13(March):1-14.

11. Jean McGowan-Jordan RJH and SM. International System for Human Cytogenomic Nomenclature 2020(ISCN). Switzerland: Karger; 2020. 1-503.

12. Ioannidis NM, Rothstein JH, Pejaver V, Middha S, McDonnell SK, Baheti S, et al. REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants. Am J Hum Genet. 2016;99(4):877-85.

13. Venselaar H, te Beek TAH, Kuipers RKP, Hekkelman ML, Vriend G. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinformatics. 2010;11:548.

14. Sue Richards, Nazneen Aziz, Sherri Bale, David Bick, Soma Das, Julie Gastier-Foster, Wayne W. Grody, Madhuri Hegde, Elaine Lyon, Elaine Spector, Karl Voelkerding and HLR. Standards and guidelines for the interpretation of sequence variants. Genet Med. 2015;17(5):405-24.

15. Rjiba K, Ayech H, Kraiem O, Slimani W, Jelloul A, Ben Hadj Hmina I, et al. Disorders of sex development in Wolf-Hirschhorn syndrome: a genotype-phenotype correlation and MSX1 as candidate gene. Mol Cytogenet. 2021;14(1):1-9.

16. Callier P, Calvel P, Matevossian A, Makrythanasis P, Bernard P, Kurosaka H, et al. Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46, XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling. PLOS Genet. 2014;10(5):1-12.

17. Juniarto AZ, van der Zwan YG, Santosa A, Ariani MD, Eggers S, Hersmus R, et al. Hormonal evaluation in relation to phenotype and genotype in 286 patients with a disorder of sex development from Indonesia. Clin Endocrinol (Oxf). 2016;85(2):247-57.

18. Wisniewski AB, Batista RL, Costa EMF, Finlayson C, Helena M, Sircili P, et al. Management of 46,XY Differences/Disorders of Sex Development (DSD) throughout Life. Endocr Rev. 2019;40(6):1547-72.

19. Abualsaud D, Hashem M, Alhashem A, Alkuraya FS. Survey of disorders of sex development in a large cohort of patients with diverse Mendelian phenotypes. Am J Med Genet Part A. 2020;1:1-12.

20. Zidoune H, Ladjouze A, Chellat-rezgoune D, Boukri A, Aman S, Teibel M, et al. Novel Genomic variants, atypical phenotypes and evidence of a digenic/oligogenic contribution to Disorders of Sex Development in a large North African Cohort. : Front Genet. 2022;13.

21. I A Hughes 1, C Houk, S F Ahmed, P A Lee LCGEC. Consensus statement on management of intersex disorders. Arch Dis Child. 2006;91(7):554-63.

22. Ahmed SF, Alimusina M, Batista RL, Domenice S, Lisboa Gomes N, McGowan R, et al. The Use of Genetics for Reaching a Diagnosis in XY DSD. Sex Dev. 2022;1-18.

23. Yu B, Liu Z, Gao Y, Wang X, Mao J, Nie M, et al. Prevalence of gene mutations in a Chinese 46, XY disorders of sex development cohort detected by targeted next-generation sequencing. Asian J Androl. 2021;2(October 2019):69-73.

24. Xue M, Wang X, Li C, Zhao M, He F, Li X. Novel pathogenic mutations in disorders of sex development associated genes cause 46,XY complete gonadal dysgenesis. Gene. 2019;718:1-11.

25. Wat MJ, Schelochkov OA, Holder AM, Breman AM, Dagli A, Bacino C, et al. Chromosome 8p23.1 deletions as a cause of complex congenital heart defects and diaphragmatic hernia. Am J Med Genet Part A. 2009;149(8):1661-77.

26. Wagner-Mahler K, Kurzenne JY, Gastaud F, Hoflack M, Panaia Ferrari P, Berard E, et al. Is interstitial 8p23 microdeletion responsible of 46, XY gonadal dysgenesis? One case report from birth to puberty. Mol Genet Genomic Med. 2019;7(3):1-9.

27. de LaPiscina IM, de Mingo C, Riedl S, Rodriguez A, Pandey AV, Fernández-Cancio M, et al. GATA4 variants in individuals with a 46, XY Disorder of Sex Development (DSD) may or may not be associated with cardiac defects depending on second hits in other DSD genes. Front Endocrinol (Lausanne). 2018;9(APR):1-10.

28. Bashamboo A, McElreavey K. Mechanism of Sex Determination in Humans: Insights from Disorders of Sex Development. Sex Dev. 2016;10(5-6):313-25.
29. Lourenço D, Brauner R, Rybczyńska M, Nihoul-Fékété C, McElreavey K, Bashamboo A. Loss-of-function mutation in GATA4 causes anomalies of human testicular development. Proc Natl Acad Sci U S A. 2011;108(4):1597–602.

30. Harris A, Siggers P, Corrochano S, Warr N, Sagar D, Grimes DT, et al. ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling. Proc Natl Acad Sci U S A. 2018;115(21):5474–9.

31. Hao HX, Xie Y, Zhang Y, Zhang O, Oster E, Avello M, et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature. 2012;485(7397):195–202.

32. Chassot AA, Gillot I, Chaboissier MC. R-spondin1, WNT4, and the ctnnb1 signaling pathway: Strict control over ovarian differentiation. Reproduction. 2014;148(6):R97-110.

33. Gonen N, Quinn A, O'Neill HC, Koopman P, Lovell-Badge R. Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone. PLoS Genet. 2017;13(1):1–17.

34. Nicol B, Yao HHC. Gonadal identity in the absence of pro-testis factor SOX9 and pro-ovary factor beta-catenin in mice. Biol Reprod. 2015;93(2):1–12.

35. Portnoi MF, Dumargne MC, Rojo S, Witchel SF, Duncan AJ, Eozenou C, et al. Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies. Hum Mol Genet. 2018;27(7):1228–40.

36. Matevossian A, Resh MD. Membrane topology of hedgehog acyltransferase. J Biol Chem. 2015;290(4):2235–43.

37. Hardy RY, Resh MD. Identification of N-terminal residues of sonic hedgehog important for palmitoylation by Hedgehog acyltransferase. J Biol Chem. 2012;287(51):42881–9.

38. Mazen I, Mekkawy M, Kamel A, Essawi M, Hassan H, Abdel-Hamid M, et al. Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development. Am J Med Genet Part A. 2021;185(6):1666–77.

39. Abdel-Salam GMH, Mazen I, Eid M, Ewida N, Shaheen R, Alkuraya FS. Biallelic novel missense HHAT variant causes syndromic microcephaly and cerebellar-vermis hypoplasia. Am J Med Genet Part A. 2019;179(6):1053–7.

**Publisher's note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

<figure><img src="image_7.png" /></figure>