SOX2 heterozygous mutation causes multiple extra-ocular phenotypes in boys

Background The SOX2 gene is widely expressed in the eyes and the central nervous system. Heterozygous mutations could cause eye malformations and hypopituitarism, and serve as the causative gene for syndromic and non-syndromic hypogonadotropic hypogonadism (HH). Our study reports three children with chromosome 46, XY, SRY (+), but SOX2 mutations. Methods Three children visited our endocrine clinic because of micropenis and/or cryptorchidism. Clinical data were collected, and one took PANEL sequencing and the others for whole exome sequencing. Then we summarized characteristics of the patients and compared with those mentioned in literature. Results Patient 1 manifested with micropenis, patient 2 with bilateral cryptorchidism and craniofacial deformities, both carrying the same reported SOX2 gene mutation (T232N), and both mutations from mothers with delayed puberty only. Patient 3 showed micropenis, mental retardation and craniofacial deformities, and the child carried a spontaneous truncation mutation (Y110X) of the SOX2 gene. This site has reported that a missense mutation caused adolescent adolescence without major eye signs. All three patients carried another gene mutations that affected hypothalamic-pituitary function: Patient 1, FGFR1 : c.238C>T/p.R80C (uncertain) from father; Patient 2, CHD7 : c.2656C>T/p.R886W (pathogenic) de novo; Patient 3, SEMA3A : c.1432G> A/p.E478K (uncertain) from mother. None had major ocular malformations, and all showed genitourinary tract malformations. Two patients had craniofacial deformities, and one patient had muscle anomality and intellectual disability. We summarized previous studies with SOX2 gene mutations and it showed: 71.2% of mutations are de novo, all patients reported whose variants inherit from parents, 15.1% parents (including mother 11.0% and father 4.1%) show completely normal phenotypes, 4.1% (3/73) variants inherit from mother with germinal mosaicism. Except for major ocular malformations (91.1%), the most common phenotype is developmental delay/mental retardation (DD/MR), accounting for 40.7%, followed by brain anomely (BA), accounting for 28.5%, male genital abnormalities (GA) for 20.3%, non-syndromic HH accounted for 4.9%, the younger the patients visit the doctor, the more common the retardation are.


Introduction
The HMG-box transcription factor SOX2 (OMIM 184429) is most notably expressed in eye, placodes, forebrain and hypothalamo-pituitary, and involved in the early embryonic development [1,2]. Loss of function mutations or deletions of SOX2 could lead to uni-or bilateral anophthalmia/microphthalmia (A / M) as well as other related disorders like anophthalmia/esophageal-genital syndrome (AEG) [3,4]. More and more studies found that SOX2 mutations could cause variable extra-ocular symptoms, including growth retardation, sensorineural hearing loss, mental retardation, intellectual disability, brain malformation, no pubertal signs and male genitourinary tract malformations (micropenis, cryptorchidism and hypospadias) [5,6]. Indeed, cases with SOX2 pathogenic mutations but no or minor ocular symptoms have been reported less frequently.
SOX2 is a highly conserved gene located in 3q26. 3-27.1. The single exon gene encodes a protein of 317 residues, which includes an N-terminal domain, a DNA-binding HMG (high-motility group) domain and a transcriptional activation domain in C-terminal [7]. At present, more than 100 cases of ophthalmology patients with A / M or other major ocular anomalies have been reported to have SOX2 mutations, mainly truncating variants caused by nonsense or frameshift [8][9][10][11][12]. SOX2 plays a pivotal role in the development of hypothalamopituitary by transactivation of mutiple genes including HESX1, and its mutations can cause hypophysial hypoplasia, reduce gonadotropin secretion, thus causing genital tract abnormalities or no puberty [2,13]. Several studies found that SOX2 heterozygous mutations cause typical signs of complete hypogonadism without major ocular malformations in men or women, say, isolated hypogonatropic hypogonadism (HH), but no another HH pathogenic gene was identified [10][11]14]. And no such Chinese patients were reported as well. Therefore, our study reported for the first time that three Chinese boys who referred due to micropenis and/or cryptorchidism, combined with craniofacial deformities or intellectual disability. The NGS found SOX2 heterozygous mutations and another HH pathogenic genes were identified. It is speculated that SOX2 and other HH pathogenic genes cooperate to cause symptoms.

Subjects And Methods
We report three cases who visited endocrinology clinic because of micropenis and/or cryptorchidism. All their phenotypes, family history, hormone levels and genetic results were collected. The characteristics of the cases were summarized and compared with those involved in the literature.
The detection method of hormones, hCG standard and prolonged stimulating test and LHRH stimulation test were referred to the published literature of our group [15][16].
NGS was completed in the Kangxu Company, patient 1 was taken panel sequencing including 167 genes involved in gonadal development, both patients 2 and 3 were carried out whole exome sequencing. DNA was extracted from peripheral blood leukocytes of patients and their parents, and the NEXTSEQ 500 sequencer (Illumina corporation, USA) was used to filter out all possible pathogenic missense, frameshift and splice site mutations. Design primers and Sanger sequencing were used to verify the mutations of samples from the same family. Mutations of MAF < 1% in East Asia people were presented here. And we checked the pathogenicity of the mutations according to ACMG.
The research protocol was approved by the Ethics Committee of the National Children's Medical Center and the Beijing Children's Hospital. Written informed consent was obtained from all participants.

Patient 1
The male patient was 6 months old at first visit, he was born at full term, and the birth weight and length were unknown. He was born with micropenis, but his intelligence and growth development were normal. He had no special facial features, no abnormalities in heart, lungs and abdomen. The penis is 2.3 cm long and 1.2 cm in diameter. The urethral meatus is normal. The volume of bilateral testis is about 2 ml. The mother had a threatened miscarriage at two months of pregnancy, and she received progesterone treatment for one month. His father's growth spurt was 17 years old, and his mother began menarche at age of 14-15 years old, and he had no family history of infertility.
The karyotype was 46, XY, and SRY was positive. Hormone levels were as follows: AMH > 23 ng/ml, INHB 126.2 pg/ml, T 434 ng dl after hCG stimulation test. LHRH stimulating test was performed in our hospital at the age of 3.5 years old. Basic LH 1.89 IU/L, FSH 5.48 IU/L, T < 20 ng/dl, peak LH/FSH = 3.47/5.48 = 0.63 > 0.6, suggesting that the pituitary has a response. MRI showed that pituitary, olfactory bulb, olfactory tract and olfactory sulcus were normal. Given the age of the patient, HH was suspected. He took testosterone undecanoate to treat micropenis for one month, then the penis was 4 cm long and 1.5 cm in diameter. The patient was followed up for 3.5 years. His hearing, smell and vision were normal, and fundus examination was normal. Later, a PANEL sequencing containing 167 gonads-associated genes was taken and found a pathogenic SOX2 varient from mother: c.695C > A/p.T232N, and an unreported pathogenic FGFR1 mutation from father: c.238C > T/p.R80C at the same time.

Patient 2
The male patient visited our clinic at the age of 2 years and 7 months. He was born at full term, with birth weight of 4 kg and a birth length of 51 cm. He was born with bilateral cryptorchidism, and had normal intelligence and growth development. Examination: eyes were normal; protruding ear, no inner cochlea, low nasal bridge, high arch bow, crooked mouth when crying, curved fifth finger, penetrating palm, but no abnormalities in heart, lungs and abdomen. The penis is 3 cm long and 1 cm in diameter. The urethral meatus is normal. The bilateral testis were located in the scrotum after surgery, and the volume was about 1 ml. No abnormalities during maternal pregnancy. The father was 172 cm, had a history of acrosomal enzyme deficiency, but the development was unknown. The mother was 170 cm, had menarche at the age of 15-16. She had one one elder sister aged 7 years, 123 cm tall, and had normal growth and development. The grandparents were consanguineous marriage. The father failed to have an IVF due to infertility, but the patient and his sister were born naturally.
The karyotype was 46, XY, and SRY was positive. Basic sex hormone levels at first visit were as follows: LH 0.12 IU / L, FSH 0.34 IU / L, T < 20 ng/dl, AMH 16.63 ng/ml, INHB 27.63 pg/ml, T 25 ng/dl after hCG prolonged test, which suggested testicular dysplasia. Now the child is 3 years and 2 months old, with normal intelligence, normal hearing, smell and vision, and no abnormalities in fundus examination. Genetic testing revealed that the patient carried both SOX2 mutation (c.695C > A/p.T232N, from mother, reported and pathogenic mutation) and CHD7 mutation (c.2656C > T/p.R886W, de novo, unreported and pathogenic mutation).

Patient 3
The male patient went to hospital because of micropenis, bilateral cryptorchidism and poor intellectural development at 5 months. Examination: sluggish face, flat pillow, flat nose, left ptosis, poor muscle strength at the back and neck, high palatal arch, decreased muscle strength of the limbs, fetal fat pad of two fingers. The penis was 1 cm long and 0.6 cm in diameter. The urethral opening was normal. Bilateral testes were located in the scrotum, the volume was about 1 ml. The patient was the first child after three gestation (one of the twins), and he was conceived through ovulation induction and artificial pregnancy. And he was born at full term, with birth weight of 3.1 kg. He could turn over at 7 months old, when he was 2 years and 8 months, he just could walk with one's help, understand simple instructions, pronounce "Mom and Dad", speak unclearly, response slowly, and he received continuous rehabilitation training. At 6 months of pregnancy, the mother had a miscarriage due to abdominal pain, and the mother didn't pregnant in next 4 years. The second pregnancy was through ovulation, but the fetus stopped developing at 2 months of pregnancy. The father had congenital scoliosis, and the development of puberty was unknown. The mother was 160 cm tall, the menarche was 13 years old, and she had polycystic ovary syndrome.
The karyotype of the patient was 46, XY, and SRY was positive. Basic hormone levels at 5 months were as follows: LH 1.4 IU/L, FSH 7.9 IU/L, T < 20 ng/dl, T 116 ng / dl after hCG stimulation test, AMH > 23 ng/ml, INHB: 74.5 pg/ml, IGF-1: 50.9 ng/ml, Peak LH/FSH = 2.59/7.9 = 0.33 < 0.6, after LHRH stimulation test, which suggested the pituitary gland could have a response. Thyroid function, ACTH and cortisol were normal. Pituitary MRI showed that the pituitary gland was normal, and the olfactory bulb, olfactory tract, and olfactory groove developed normally. The boy received testosterone undecanoate treatment for micropenis. The child was 3 years old nowadays, but he couldn't walk steadily, catching up with mental and physical development, and is unstable. He was 88.3 cm tall, and 2 cm below the 3rd percentile of the growth curve. After treatment with testosterone undecanoate, the penis growed to 3.5 cm long and 1.1 cm in diameter. Hearing, smell and vision were normal, and no abnormalities were found on fundus examination. Genetic testing revealed that the patient harbored both SOX2 mutation (c.330C > A/p.Y110X, de novo, unreported and pathogenic mutation) and SEMA3A mutation (c.1432G > A/p.E478K, from mother, unreported, and clinical significance is uncertain).
In our single centre, more than 800 patients with 46, XY DSD received genetic tests, 118 patients were suspected with HH among these, and SOX2 gene mutation accounted for 2.5% of the 118 patients. Patient 1 showed only micropenis, without any deformity and mental and physical development disorders, while patient 2 carrying the same SOX2 gene mutation manifested with bilateral cryptorchidism and craniofacial deformities. Their mothers are carriers of SOX2 mutation, and both only show delayed menarche. Patient 3 showed micropenis, bilateral cryptorchidism, mental retardation, eye and craniofacial deformities, see Tables 1, 2 and Fig. 1 for details. Patient 1 and 2 carry the same reported pathogenic site mutation (p.T232N) of the SOX2 gene, which is located in the carboxy terminal transcription activation region, and Patient 3 carries a nonsense mutation (p.Y110X ), Close to the HMG region, which has not been reported in the literature, but there are reports of the same site missense pathogenic mutation (p.Y110C). Intersting, we found that all three cases carry another gene varient that caused HH meanwhile, see Fig. 1 for the combined gene mutation.

Literature summary and comparison with our patients
In 2003, the SOX2 gene mutation was first reported in patients with A/M deformities. Up to now, a total of 123 cases have been reported, including male (57/109, 52.3%) and female (52/109, 47.7%), some are not provided, visiting age ranges from fetus to 65 years old, 43.9% (25/57) male patients showed genital abnormalities, see supplementary table 1 and Table 3. Almost all patients choose to ophthalmology clinic, except for those patients without major eye deformities. While all three patients in our study are male, and show no major ocular symptoms.
Among those patients, 91.1% of patients have major ocular deformities. All patients reported whose variants inherit from parents, 15.1% parents (including mother 11.0% and father 4.1%) show completely normal phenotypes, 4.1% (3/73) variants inherit from mother with germinal mosaicism. Among the extraocular symptoms, the most common is developmental delay/mental retardation (DD/MR), accounting for 40.7%, followed by brain anomely (BA), accounting for 28.5%, motor development delay (MD) for 22%, male genital abnormalities (GA, including micropenis, cryptorchidism and hypospadias) for 20.3%, short stature (SS) for 17.1%, facial dysmorphism (FD) accounting for 12.2%, non-syndromic HH accounted for 4.9%, including three male patients (2.4%). Clinical and genetic features of all patients without major ocular deformities were listed in Table 4. The three patients in the study were mainly genital abnormalities, left ptosis, hypotonia, short stature, and intellectual impairment, but with no major ocular abnormalities. No seizures were found.

Discussion
The unilateral or bilateral A/M itself has a low prevalence rate. Among them, the detection rate of SOX2 gene heterozygous mutations is about 10-20% [9], and some patients are accompanied by extra-ocular symptoms, including growth retardation, sensorineural hearing loss, intellectual impairment, corpus callosum dysplasia, pubertal dysplasia, male external genital malformation (micropenis, cryptorchidism and hypospadias) [36]. However, there are still few reports of patients with the gene variants who have no or minor eye symptoms.

Phenotypes caused by SOX2 mutations range from normal to severe ocular symptoms
Totally 91.1% patients show A/M or severe coloboma in all cases reported with SOX2 mutatitons. All patients reported whose variants inherit from parents, 15.1% parents (including mother 11.0% and father 4.1%) show completely normal phenotypes, 4.1% (3/73) variants inherit from mother with germinal mosaicism, both children in 3 families [21,23,24] have major ocular deformities, while their mothers manifest normal phenotypes. Table 4, we present 11 cases with SOX2 mutations but without major ocular deformities. Of our more than 500 genetically positive 46, XY DSD cases in our single centre, only 3 patients (0.6‰) were reported in the study, suggesting that the incidence of SOX2 gene mutations is lower in patients without major ocular deformities. In our study, we reports 3 patients with micropenis, cryptorchidism and/or hypospadias as the main phenotypes but no significant ocular deformity. It is noteworthy that both patients 1 and 2 carry the same mutation of SOX2 gene (c.695C > A / p.T232N) transmitted from the mother, but the phenotypes are very different. Patient 1 shows merely micropenis, while patient 2 manifested bilateral cryptorchidism, facial deformities and crooked mouth crying syndrome, but both had normal vision and no ocular deformities, and their mothers have delayed menarche and no other phenotypes. Crooked mouth crying syndrome could manifest as deformities of eyes and ears and congenital heart disease, but it often cause by a minor deletion of chromosome 22q11, therefore, phenotypes of patient 2 couldn't be explained by the syndrome. Previous study report that one Chinese father and son carrying the same SOX2 mutation as our patients, the father showed ocular defects but no reproductive system abnormalities, the son also showed ocular defects, arachnoid cysts and penoscrotal hypospadias, but no follow-up description of pubertal development and fertility [37].

And in
In another family report containing a frameshift mutation in the SOX2 gene (p.G280Afs91X), the mother was diagnosed with IHH due to primary amenorrhea and no secondary sexual development at the age of 18, without ocular diseases or other deformities. With the help of assisted reproductive fertility, she rears one son and one daughter, the son shows anophthalmia, and the daughter has unilateral microphthalmia deformity. All of these suggest that patients with SOX2 mutations have a broad phenotype spectrum, and there is no obvious correlation between genotypes and phenotypes [9,38].

SOX2 may cooperate with other pathogenic genes associated with hypothalamic-pituitary axis to cause HH
Heterozygous SOX2 mutations in human patients commonly cause pituitary hypoplasia on imaging, usually leading to low concentrations of LH and FSH (that is, typical HH), or GH deficiency and short stature in some conditions. However, HH was also observed in patients without pituitary hypoplasia, further study showed that SOX2 mutations could reduce GnRH numbers and misdirect axonal projections, as evidenced by the phenomenon that HH patients carrying SOX2 mutations could respond to GnRH stimulation. Our previous study and others showed multiple gene defects might synergize to cause a more severe HH phenotype in at least 20% of cases.
Heterozygous mutations in the SOX2 gene could lead to syndromic HH with A/M/coloboma [4,5,9]. Since 2003, only 6 cases (4.9%) of patients with non-syndromic HH (except for absense of puberty, some patients also show micropenis and/or cryptorchidism) have been reported [8,11,14,30,[38][39], while micropenis and cyptorchidism are the main clues of HH during childhood. The 3 patients in our study are currently young and need further follow-up. However, considering the symptoms, signs and genetic results, they are highly suspected diagnosis of IHH (including patient 2 with crooked mouth crying syndrome), which suggests that phenotypes causing by SOX2 mutations can be various.
However, previous reports have not detected or only detected a small part of the HH pathogenic genes, therefore, only the SOX2 gene mutation was found to be the causative gene for non-syndromic HH. According to previous reports, the SOX2 gene heterozygous pathogenic mutations could cause related malformations in the study [40], but the specific mechanism and whether it may be synergistic with another HH gene still need further study. Our study provided clues for the different phenotypes couldn't be ruled out as the result of oligogenic genetic architecture. Patient 2 carried both SOX2 and CHD7 gene mutation simultaneously, T was still low after the hCG prolonged test, and the INHB level was low, suggesting that the patient may had testicular Leydig cells and Sertoli cells dysplasia. One study has found that SOX2 and CHD7 could form a complex, and cooperatively activate downstream genes (such as Notch and Shh pathway genes) to participate in the development and maturation of testicular cells [41]. It is speculated that the SOX2 and CHD7 mutations in this patient may aggravate the impact of a single gene mutation on testicular function, which needs to be confirmed by further functional studies. Patient 3 carried a new nonsense mutation (p.Y110X) of SOX2 gene, and he showed micropenis, cyptorchidism and mental retardation but normal vision. In 2014, Takagi et al. reported a missense mutation (p.Y110C) of the SOX2 gene at this site, the patient presented with micropenis and no pubertal development at the age of 20, he began to appear generalized seizures at the age of 3, and received right retinal detachment surgery at the age of 14 years old. Y110 is a critical amino acid near the DNA-binding HMG region. Mutation attenuates the activation of the downstream target gene HESX1 [30]. While the patient in our study occurred a nonsense mutation at this site, and it's speculated that the mutation would have a greater impact on target gene activation and subsequent gonadotropin levels.

Conclusion
SOX2 mutations could cause a broad phenotype spectrum from completely normal to severe ocular malformations, retardation and most are de novo. Except for major ocular malformations and retardation, GA/HH is another common symptom. GA/HH may be the only symptom, and SOX2 may cooperate with another HH pathogenic genes to cause non-syndromic HH.

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