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Accepted Manuscript
Increased transactivation and impaired repression of β-catenin-mediated transcription
associated with a novel SOX3 missense mutation in an X-linked hypopituitarism
pedigree with modest growth failure
Tingting Yu, Guoying Chang, Qing Cheng, Ruen Yao, Juan Li, Yufei Xu, Guoqiang Li,
Yu Ding, Yanrong Qing, Niu Li, Yiping Shen, Xiumin Wang, Jian Wang
PII:
S0303-7207(18)30241-7
DOI:
10.1016/j.mce.2018.08.006
Reference:
MCE 10284
To appear in:
Molecular and Cellular Endocrinology
Received Date: 14 April 2018
Revised Date:
14 August 2018
Accepted Date: 15 August 2018
Please cite this article as: Yu, T., Chang, G., Cheng, Q., Yao, R., Li, J., Xu, Y., Li, G., Ding, Y., Qing, Y.,
Li, N., Shen, Y., Wang, X., Wang, J., Increased transactivation and impaired repression of β-cateninmediated transcription associated with a novel SOX3 missense mutation in an X-linked hypopituitarism
pedigree with modest growth failure, Molecular and Cellular Endocrinology (2018), doi: 10.1016/
j.mce.2018.08.006.
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ACCEPTED MANUSCRIPT
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Increased transactivation and impaired repression of β-catenin-mediated
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transcription associated with a novel SOX3 missense mutation in an X-linked
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hypopituitarism pedigree with modest growth failure
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Tingting Yu1,*, Guoying Chang2,*, Qing Cheng2,*, Ruen Yao1, Juan Li2, Yufei Xu1,
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Guoqiang Li1, Yu Ding2, Yanrong Qing1, Niu Li1, Yiping Shen1,3, Xiumin Wang2,#,
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Jian Wang1,#
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Department of Medical Genetics and Molecular Diagnostic Laboratory, Shanghai
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Children’s Medical Center, Shanghai Jiaotong University School of Medicine,
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Shanghai, China
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Shanghai Jiaotong University School of Medicine, Shanghai, China
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3
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Massachusetts, USA.
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Department of Endocrinology and Metabolism, Shanghai Children’s Medical Center,
Division of Genetics and Genomics, Boston Children’s Hospital, Boston,
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*
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authors.
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These authors contributed equally to this work, and should be considered as co-first
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Wang (labwangjian@shsmu.edu.cn), Shanghai Children’s Medical Center, Shanghai
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Jiaotong University School of Medicine. 1678 Dongfang Road, Shanghai 200127, P.R
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China; Fax 86-21-58756923.
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Corresponding author: Dr. Xiumin Wang (wangxiumin1019@126.com) and Dr. Jian
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Abstract
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SOX3, a transcription factor of the SRY-related high mobility group box family, has
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been implicated in the etiology of X-linked hypopituitarism. Here, we report a
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Chinese pedigree of X-linked hypopituitarism with variable phenotypes. Despite the
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complete growth hormone deficiency, the growth failure of the patients was relatively
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modest. A rare point variant of SOX3 (c.424C>A; p.P142T) was identified in the
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pedigree via target panel sequencing. An in vitro study showed that both the
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expression and nuclear targeting of SOX3 remained unaffected by the variant.
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However,
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β-catenin-mediated transcription were noticed as a result of the SOX3 variant. This is
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the first study to report that the rare SOX3 missense variant associated with
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hypopituitarism possibly due to increased activation of SOX3 target genes and
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disregulation of β-catenin target genes. In addition, we have expanded the phenotypic
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spectrum associated with SOX3 mutations.
transcriptional
impaired
repression
of
Key words: congenital hypopituitarism; growth hormone; SOX3; mutation
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and
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activation
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increased
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Abbreviations: IGHD, isolated growth hormone deficiency; CPHD, combined
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pituitary hormone deficiency; aa, amino acids; HMG, high mobility group; GH,
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growth hormone; SCMC, Shanghai Children’s Medical Center; IVF, in vitro fertilized;
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SDS, standard deviation score; BMI , body mass index; TSH, thyroid-stimulating
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hormone; ACTH, adrenocorticotropic hormone; IGF-I, insulin-like growth factor I;
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IGFBP-3, insulin-like growth factor-binding protein 3; LH,
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FSH, follicle-stimulating hormone; MRI, magnetic resonance imaging; GnRH,
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gonadotropin-releasing hormone; OGTT, oral glucose tolerance test; NGS, next
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generation sequencing; PCR, polymerase chain reaction; WT, wild type; HEK, human
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embryonic kidney; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified
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Eagle's medium; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride.
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2
luteinizing hormone;
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1. Introduction
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Hypopituitarism is a disorder characterized by the diminished production of pituitary
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hormones. It was first described in 1914 (Simmonds, 1914) and includes isolated
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hormone deficiency such as isolated growth hormone deficiency (IGHD), combined
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pituitary hormone deficiency (CPHD), and panhypopituitarism (Alatzoglou et al.,
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2014; Castinetti et al., 2015). Acquired hypopituitarism occurs in all age groups due to
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various causes including pituitary tumor, meningitis, and traumatic brain injury,
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whereas congenital events usually present in the neonatal period or somewhat later in
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life. Congenital hypopituitarism affects about 1 in 10,000 children (de Moraes et al.,
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2012) and is caused by defects in the genes involved in the development of either the
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pituitary gland or the hypothalamus. Numerous genetic alterations have been reported
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to be responsible for congenital hypopituitarism (Fang et al., 2016; Romero et al.,
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2009; Stieg et al., 2017).
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SOX3 is a single-exon gene located on the X chromosome at position q27.1. The
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encoded protein (SOX3) has a length of 446 amino acids (aa) and belongs to the
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SRY-related high mobility group (HMG) box (SOX) family of transcription factors.
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SOX3 consists of a short N-terminal domain, a HMG DNA binding domain, and a
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long C-terminal domain, which contains four poly-alanine tracts (Mojsin et al., 2010;
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Stevanoviä
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development (Rizzoti et al., 2004). The involvement of SOX3 with congenital
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hypopituitarism was first described in 2002, when Laumonnier et al. reported an
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in-frame duplication of 33 bp in the first poly-alanine tract of SOX3 in a patient with
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IGHD and mental retardation (Laumonnier et al., 2002). Since then, several other
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insertions and deletions of the poly-alanine tracts of SOX3 have been detected in
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hypopituitarism patients (Alatzoglou et al., 2011; Burkitt Wright et al., 2009; Takagi
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et al., 2014; Woods et al., 2005). Moreover, duplications of fragments containing
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SOX3 have also been identified in individuals with hypopituitarism (Bauters et al.,
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2014; Rosolowsky et al., 2015).
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et al., 1993). It plays a key role during the early stages of pituitary
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In this study, we present a Chinese pedigree with congenital hypopituitarism. Variable
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phenotypes including complete growth hormone (GH) deficiency, structural defects of
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the pituitary, callosal abnormalities, hypoplastic genitalia, mild learning difficulty,
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astigmatism, and myopia were noticed in two affected male siblings. The growth
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failure of the patients was relatively modest. SOX3 c.424C>A; p.P142T were
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identified in the pedigree via targeted panel sequencing of the 2,742 genes known to
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cause inherited disorders. In vitro studies were further performed to evaluate the
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functional impact of the variant on SOX3 and its transcriptional and downstream
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signaling activities.
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2. Materials and Methods
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2.1. Case report
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Two male twins (III3 and III4) with an age of 11 years 2 months were referred to the
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pediatric endocrine department of Shanghai Children’s Medical Center (SCMC) with
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a main complaint of poor growth velocity during the preceding two years. They were
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in vitro fertilized (IVF) twins and were born at full term via Cesarian section to
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non-consanguineous Chinese parents. The birth weights of III3 and III4 were 3250 g
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[-0.17 standard deviation score (SDS)] and 3150 g (-0.43 SDS), and their lengths at
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birth were 50 cm (-0.2 SDS) and 48 cm (-1.3 SDS), respectively. Before their birth,
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the couple had undergone two spontaneous abortions (Figure 1a).
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III3 showed a slow growth velocity from 9 to 11 years old (4 cm/year, shown in
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Figure 1b) and when he was referred to us, his voice was already broken. Upon
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physical examination, his height was 143.0 cm (-0.5 SDS), weight 43.0 kg, and body
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mass index (BMI) 21.0 kg/m2. The pubic hair Tanner stage was II. The testicular
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volume is 10 ml per testis. And the stretched penile length was 4 cm. Laboratory
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investigations showed levels within the reference ranges including lipids, fasting
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glucose, glycosylated hemoglobin, prolactin, thyroid hormone, thyroid-stimulating
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hormone
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17-hydroxyprogesterone. The level of insulin-like growth factor I (IGF-I) was 80.8
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(TSH),
cortisol,
adrenocorticotropic
4
hormone
(ACTH),
and
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ng/ml (< -2 SD) and that of insulin-like growth factor-binding protein 3 (IGFBP-3)
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was 41 ug/ml (between -2 SD and -1 SD). Due to the low level of IGF-1, a further GH
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stimulation test was conducted and the result showed a complete GH deficiency (peak
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GH response to clonidine: 0.42 ng/ml; arginine: 0.26 ng/ml). The serum luteinizing
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hormone (LH), follicle-stimulating hormone (FSH), and testosterone were at the level
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of puberty (3.13 mIU/ml, 3.28 mIU/ml, and 3.35 ng/ml, respectively). His bone age
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was that of an 11-year-old compared to the Greulich and Pyle atlas. Ultrasound for
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abdomen and thyroid were normal. The right testicle was 32×15×22 mm and the left
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was 29×14×24 mm. Cerebral magnetic resonance imaging (MRI) showed a small
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anterior pituitary gland, a hypoplastic pituitary stalk, and an ectopic posterior pituitary
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(Figure 2a).
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III4 was born with bilateral cryptorchidism and received orchiopexy at the age of 5
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years. The growth velocity was 2.5 cm/year during the preceding two years (Figure
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1b). His height was 140 cm (-0.9 SDS), weight 45 kg, and BMI 22.9 kg/m2. The pubic
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hair Tanner stage was I. The volumes of the bilateral testes were 2 ml, respectively.
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And the stretched penile length was 2 cm. Laboratory tests showed low plasma
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concentrations of IGF-1 (40.3 ng/ml; < -2 SD) and IGFBP-3 (2.20 ug/ml; < -2 SD). A
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GH stimulation test indicated complete GH deficiency (peak GH response to
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clonidine: 0.08 ng/ml; arginine: 0.11 ng/ml). The patient showed a prepubertal
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response to the gonadotropin-releasing hormone (GnRH) stimulation test (Table 1)
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and the level of testosterone was low (< 0.10 ng/ml). Oral glucose tolerance test
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(OGTT) indicated mild insulin insensitivity (Table 2). Other biochemical and
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endocrine tests were normal. The X-ray indicated a bone age of 11-year-old.
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Abdominal ultrasound revealed fatty infiltration of the liver. The right testicle was
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32×15×22 mm and the left was 29×14×24 mm. Brain MRI revealed anterior pituitary,
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pituitary stalk hypoplasia, ectopic posterior pituitary, corpus callosum hypogenesis,
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and midline lipoma (Figure 2b).
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Mild learning difficulties were noted in both siblings. However, their intelligence
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quotients were not measured. Astigmatism and myopia were detected in both III3 and
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III4 since the age of 6 years. No other abnormalities were found and their facial
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appearance was normal. There was no family history of note. The mother had one
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healthy brother and none of her known male relatives had similar problems compared
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to her sons.
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Following confirmation of the clinical diagnosis of GH deficiency, the use of GH was
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recommended in these two patients (0.15 U/kg/d). Both showed an excellent response
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to GH therapy, with the growth velocity increasing to about 10 cm/year (Figure 1b).
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The heights of III3 and III4 at an age of 12 years 7 months were 157.0 cm (0.09 SDS)
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and 155.0 cm (-0.8 SDS), respectively. The blood levels of IGF-1 were significantly
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increased (632 ng/ml in III3 and 611 ng/ml in III4 after 3 months of treatment). In
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addition, we also suggested controlling their weight. The index of glucose and
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glycosylated hemoglobin was normal during follow-up. The study was approved by
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the ethics committee of the SCMC. Informed consent was obtained from the parents
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of the children described in the study.
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2.2. Target sequencing and variant verification
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Genomic DNA was extracted from peripheral blood samples of both the siblings and
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their parents using the Gentra Puregene Blood Kit (Qiagen, Hilden, Germany)
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according to the manufacturer’s protocol. Next generation sequencing (NGS) was
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performed on the III4 to screen for causal variants. Briefly, 3 µg DNA was sheared to
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create fragments of 150 to 200 bp in size. An adaptor-ligated library was prepared
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using the SureSelectXT Library Prep Kit (Agilent Technologies, Santa Clara, CA,
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USA). 2,742 genes known to cause inherited disorders were captured using the
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ClearSeq Inherited Disease panel kit (Agilent Technologies). NGS was performed on
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an Illumina Hiseq X Ten System (Illumina, San Diego, CA, USA).
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Base calling and quality assessments of sequence reads were performed using the
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Illumina Sequence Control Software with Real Time Analysis. Paired end reads were
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aligned to the GRCh37/hg19 human reference sequence using the NextGENe
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software (SoftGenetics, State College, PA, USA). All variants were saved in VCF
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format and uploaded to the Ingenuity Variant Analysis (Ingenuity Systems, Redwood
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City, CA, USA) for biological analysis and interpretation. Variants reported in public
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databases (i.e., the Genome Aggregation Database, Exome Aggregation Consortium,
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NHLBI Exome Sequencing Project, and 1000 Genomes Project) were used as
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frequency filter.
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The exon of SOX3 was amplified via polymerase chain reaction (PCR) from the
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genomic DNA of both the siblings and their parents (primer sequence available upon
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request). Sanger sequencing using an ABI 3700 sequencer (Applied Biosystems,
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Foster City, CA, USA) was performed to analyze PCR products.
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2.3. In silico analysis of the SOX3 p.P142T variant
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The conservation analysis of the SOX3 P142 was run by MegAlign. The potential
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pathogenicity
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(http://genetics.bwh.harvard.edu/pph2/), PROVEAN (http://provean.jcvi.org/genome
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_submit_2.php?species?human), SIFT (http://sift.Jcvi.org/), and MutationTaster (http:
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//www.mutationtaster.org/ChrPos.html). Homology modeling of the p.P142T mutant
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SOX3
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Oct1.Sox2.Hoxb1-DNA ternary transcription factor complex structure serving as
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template (Protein Data Bank entry 1O4X) (Williams et al., 2004).
SOX3
variant
was
predicted
via
PolyPhen-2
was
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using
PyMOL
open-source
software
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the
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2.4. Plasmids and cloning
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To generate the Flag-tagged wild type (WT) and p.P142T mutant SOX3 expression
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vectors, the SOX3 coding region was amplified from the DNA of the siblings’ mother
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using the forward primer 5’-acaaggacgatgatgacaagggatccATGCGACCTGTTCGAGA
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GAA-3’ and the reverse primer 5’-cagtgtgatggatatctgcagaattcGGTGCTCAGATGTG
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GGTCA-3’. The PCR products were further cloned into pcDNA3.1-Flag vector using
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a Quick fusion cloning kit (Bimake, Houston, TX, USA), following the
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manufacturer’s protocol. A 944-bp HESX1 promoter sequence (-620 to +324 bp) was
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amplified from human genomic DNA and cloned into the pGL3-basic vector
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(Promega, Madison, WI, USA) to generate a luciferase reporter plasmid (HESX1-luc).
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The TOPFLASH reporter plasmid and the human β-catenin expression construct were
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generously provided by Dr. Ping Wang (School of Medicine, Tongji University,
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China). All vectors were confirmed via DNA sequencing.
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2.5. Cell culture and transient transfection
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Human embryonic kidney (HEK) 293T cells and Chinese hamster ovary (CHO) cells
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were obtained from the ATCC (American Type Culture Collection, Manassas, VA,
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USA) and maintained in Dulbecco's modified Eagle's medium (DMEM),
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supplemented with 10% (v/v) fetal bovine serum (FBS) in a humidified atmosphere of
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5% CO2 atmosphere at 37°C. Transfection was performed using the jetPRIME
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transfection reagent (Polyplus Transfection, Illkirch, France) according to the
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manufacturer's instructions.
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2.6. Western blot analysis
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HEK 293T cells were seeded (6×105 cells/well) into 12-well plates and transfected
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with 1 μg of the SOX3 WT construct, the SOX3 p.P142T mutant construct, and an
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empty backbone plasmid, respectively. Proteins collected from the whole cell lysates
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24 h post-transfection were separated on 10% SDS-PAGE gels, transferred to
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polyvinylidene difluoride (PVDF) membranes, and probed with mouse anti-Flag
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monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). β-actin was used as
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control for consistent loading. Images were analyzed and quantified using ImageJ
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(Schneider et al., 2012).
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2.7. Immunofluorescence staining
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CHO cells (2×104 cells/well) were grown on cover slips in a 24-well plate and
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transfected with 0.5 μg of the SOX3 WT construct, the SOX3 p.P142T mutant
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construct, and an empty backbone plasmid, respectively. 24 h after transfection, the
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cells were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100 in
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PBS, and blocked in 1% BSA. Immunostaining was performed using mouse anti-Flag
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monoclonal antibody (Sigma-Aldrich) incubation for 1 h followed by labelling with
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Alexa Fluor® 647 conjugated anti-mouse IgG (Cell Signaling Technology, Danvers,
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MA, USA) for 1 h. The cover slips were then mounted on microscope slides using
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mounting medium with DAPI (Thermo Fisher Scientific, Waltham, MA, USA) and
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analyzed using a Leica DM6000 fluorescence microscope (Leica Microsystems,
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Wetzlar, Germany).
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2.8. Luciferase assays
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The regulatory role of SOX3 in modulating the transcription was monitored using the
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HESX1-luc plasmid. In brief, HEK 293T cells were seeded into 96-well plates at a
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density of 6×104 cells per well and incubated for 24 h. The cells were co-transfected
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with 39 ng Hesx1-luc, 1 ng pRL-SV40, and increasing quantities of SOX3 expression
254
vectors (20 ng, 100 ng and 160 ng, respectively). The total amount of transfected
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DNA was normalized to 200 ng by addition of the appropriate amount of empty
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expression vector. Luciferase activity was measured at 24 h post-transfection with the
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Dual-Glo Luciferase assay system (Promega) and normalized to renilla luciferase,
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encoded by pRL-SV40. For the TOPFLASH assay, HEK 293T cells were
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co-transfected with 39 ng TOPFLASH reporter, 1 ng pRL-SV40, 20 ng β-catenin
260
expression construct, and increasing amounts of SOX3 expression vectors (5 ng and
261
10 ng, respectively). The total amount of transfected DNA was normalized to 100 ng.
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An appropriate amount of empty backbone plasmid was used whenever necessary.
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Luciferase activity was determined identical to the transcription assay. Biological and
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technical triplicates were performed.
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2.9. Statistical analysis
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Comparisons were made using two-tailed Student’s t tests. Results are shown as
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means ± SD (n = 3). Values of p < 0.05 were considered significant.
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3. Results
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3.1. Identification of a novel SOX3 variant in a Chinese pedigree with X-linked
272
hypopituitarism
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Target sequencing of III4, followed by bioinformatics analysis, filtering against public
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databases, and biological analysis showed a missense variant in SOX3, i.e. SOX3
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c.424C>A; p.P142T. This variant has not been previously reported in the Human
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Genome Mutation Database (http://www.hgmd.cf.ac.uk) nor in control databases such
277
as the Genome Aggregation Database (http://gnomad.broadinstitute.org/), Exome
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Aggregation Consortium (http://exac.broadinstitute.org), NHLBI Exome Sequencing
279
Project (http://evs.gs.washington.edu/EVS), or the 1000 Genomes Project (http://www.
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1000genomes.org); this variant is therefore novel. The variant was further confirmed
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using Sanger sequencing in the pedigree. III3 also harbored the same SOX3 missense
282
variant. It was detected in their mother at the heterozygous state, while the father was
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wild-type (Figure 1c).
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3.2. In silico analysis of the SOX3 p.P142T variant
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The SOX3 P142 residue is highly conserved not only in different species (Figure 3a),
287
but also among the amino acid sequences of various human SOX transcription factors
288
(Figure 3b). It is located in the N-terminal tail of the HMG domain of SOX3, which
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binds to the minor groove and bends DNA (Figure 3c). The substitution of proline to
290
threonine at the position 142 was predicted to have a deleterious effect on the SOX3
291
product by multiple in silico predictive algorithms including MutationTaster (disease
292
causing), PolyPhen-2 (probably damaging), PROVEAN (deleterious), and SIFT
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(damaging).
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3.3. SOX3 p.P142T variant showed no alteration in SOX3 expression
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The effects of p.P142T variant on SOX3 protein synthesis and sub-cellular
297
localization were determined via western blot assay and immunofluorescence staining.
298
No significant difference was observed in the expression level and molecular weight
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between the wild type and p.P142T mutant SOX3 via western blot (Figure 4).
300
Immunofluorescence staining showed that both the wild type and p.P142T mutant
301
SOX3 localized in the nucleus (Figure 5).
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3.4. The SOX3 p.P142T variant resulted in increased transcriptional activity
304
To evaluate the impact of the p.P142T variant on SOX3 transcriptional activity, a
305
luciferase assay was performed using a reporter construct containing a fragment of
306
HESX1 promoter. Both SOX3 WT and p.P142T variant stimulated the transcription of
307
the HESX1 reporter in a dose-dependent manner. SOX3 WT activated the HESX1
308
reporter up to approximately four-fold. In contrast, the SOX3 p.P142T mutant
309
exhibited significantly increased transcriptional activity, which activated the HESX1
310
reporter up to eight-fold (Figure 6a).
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3.5. The
p.P142T
variant
exhibited
reduced
repression
of
313
β-catenin-mediated transcription
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The regulatory role of SOX3 in modulating the β-catenin-mediated transcription was
315
monitored using TOPFLASH assay. Constitutive activation of the TOPFLASH
316
reporter construct induced by β-catenin was observed in HEK 293T cells. The
317
activation was suppressed by both SOX3 WT and the SOX3 p.P142T mutant in a
318
dose-dependent manner. However, compared to SOX3 WT, the SOX3 p.P142T
319
mutant showed less than two to three fold repression of β-catenin-mediated
320
transcription (Figure 6b).
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SOX3
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4. Discussion
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Pituitary development is a complex process that requires the sequential temporal and
324
spatial expression of a cascade of transcription factors and signaling molecules. SOX3
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is one of the transcription factors involved in the early development of the pituitary
326
and has been implicated in the etiology of X-linked hypopituitarism. Here, we report a
327
new pedigree with X-linked hypopituitarism and identified the novel SOX3 missense
328
variant
c.424C>A;
p.P142T.
According
11
to
previous
literature,
X-linked
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hypopituitarism patients with SOX3 alterations have highly variable phenotypes
330
(Alatzoglou et al., 2011; Bauters et al., 2014; Burkitt Wright et al., 2009; Izumi et al.,
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2014; Laumonnier et al., 2002; Takagi et al., 2014; Woods et al., 2005). The
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phenotypic heterogeneity was also noticed in our patients even with an identical SOX3
333
mutation. The affected III3 presented with decreased growth velocity, mild learning
334
difficulty, astigmatism, and myopia. Structural defects of the pituitary were observed
335
via brain MRI, indicating failure of normal pituitary development. The patient was
336
diagnosed with IGHD based on the markedly reduced serum concentrations of the GH
337
during the stimulation tests, low levels of serum IGF-I and IGFBP-3, and normal
338
concentrations of other pituitary hormones. However, III4 presented with a more
339
complicated phenotype. In addition to the features found in III3, III4 also had
340
abnormal midline structures such as corpus callosum hypogenesis and midline lipoma.
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Hypoplastic genitalia including bilateral cryptorchidism, small testicles, and
342
micropenis were also noted in III4. The patient presented a prepubertal stage
343
according to GnRH stimulation test. Whether III4 will develop gonadotropin
344
deficiency and hypogonadotropic hypogonadism requires further follow-up.
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Patients of SOX3-associated hypopituitarism typically present short stature and
347
delayed bone age. However, the growth failure of the affected twins was relatively
348
modest and their bone ages were normal. Similar situations have occasionally been
349
described in patients with SOX3 involvement (Burkitt Wright et al., 2009; Woods et
350
al., 2005). Linear growth is a multifactorial trait, influenced by the interaction of
351
genetic, nutritional, hormonal, and environmental factors (Hathout et al., 1999; Lazar
352
et al., 2003). To evaluate the genetic impact on the growth, we analyzed the target
353
sequencing data of III4. 20 variants in genes related to short stature and 1 variant
354
related to tall stature were detected (see supplemental table 1). However, most of the
355
variants were rare polymorphisms or functionally predicted to be benign by various in
356
silico predictive algorithms. Obesity-induced hyperinsulinemia, hyperprolactinemia,
357
elevated leptin levels, bioavailable GH/IGF-I variants, and unknown growth factors
358
with sufficient compensatory ability have been offered as an explanation for the linear
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growth independent of GH (Hathout et al., 1999; Makras et al., 2004; Maor et al.,
360
2002). However, its exact mechanism has not been elucidated to date. Our patients
361
were not obese and their prolactin levels were within the normal range, yet the insulin
362
level of III4 was elevated as indicated via OGTT. Insulin is a stimulatory factor
363
exerting a mitogenic effect. The elevated insulin level may contribute to the
364
maintained growth in III4. However, the insulin level was unknown in III3 since he
365
refused to take part in the OGTT. Moreover, the blood levels of GH and IGF-I had
366
never been measured in the siblings prior to the age of 11. Whether the growth before
367
the age of 11 was induced by normal or near-normal levels of GH and IGF-I remains
368
unknown.
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Via phenotypic relevance analysis, functional prediction, and genetic pattern analysis,
371
SOX3 c.424C>A; p.P142T was further identified as potentially causative. Both large
372
or submicroscopic duplications encompassing SOX3 and mutations within SOX3 have
373
been found in individuals with hypopituitarism (Bauters et al., 2014; Rosolowsky et
374
al., 2015; Solomon et al., 2004; Stagi et al., 2014; Stankiewicz et al., 2005; Woods et
375
al., 2005). Most SOX3 mutations are insertions and deletions in the first polyalanine
376
tract (Alatzoglou et al., 2011; Laumonnier et al., 2002; Takagi et al., 2014).
377
Pathogenic point mutations of SOX3 seem to be rare. Two point variations of SOX3
378
reported in hypopituitarism cases are SOX3 c. 14G>A; p.R5Q and SOX3 c.449C>A;
379
p.S150Y (Alatzoglou et al., 2011; Jelsig et al., 2018). SOX3 p.R5Q has been described
380
in a boy who presented with CPHD (GH, LH, and FSH deficiency) (Alatzoglou et al.,
381
2011). However, it seems to be a benign or likely benign variation since in vitro study
382
showed no functional effect of p.R5Q on SOX3 (Alatzoglou et al., 2011). SOX3
383
p.S150Y was recently detected in a family with three affected males with several
384
clinical
385
microphthalmia, coloboma, facial dysmorphology, and dental anomalies (Jelsig et al.,
386
2018). This has been predicted to be disease causing by various software packages
387
(Jelsig et al., 2018). However, no functional studies were performed on SOX3
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features
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p.S150Y to date.
389
In mice, Sox3 is highly expressed in the ventral hypothalamus and infundibulum
391
(Rizzoti et al., 2004). It is essential for the formation of the hypothalamo-pituitary
392
axis. Disruption of Sox3 in mice leads to hypopituitarism, craniofacial abnormalities,
393
and midline central nervous system (CNS) defects (Rizzoti et al., 2004). In humans,
394
polyalanine tract expansions of SOX3 result in reduced transactivation with
395
aggresome formation or impaired nuclear localization of the mutant protein (Wong et
396
al., 2007; Woods et al., 2005). In contrast, polyalanine tract deletions are associated
397
with increased transcriptional activation (Alatzoglou et al., 2011; Takagi et al., 2014).
398
The existing studies indicate that pituitary development may be very sensitive to
399
SOX3 dosage.
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To evaluate the impact of SOX3 c.424C>A; p.P142T on the resulting protein, in silico
402
analysis and in vitro study were conducted. SOX3 p.P142 is highly conserved and
403
located in the HMG DNA binding domain. Proline, a nonpolar aliphatic amino acid
404
with a distinctive non-reactive side chain, is essential for the conservation of a
405
particular protein fold. The substitution of proline with threonine at the position 142
406
may affect the conformation and alter its DNA-binding properties. Furthermore, the
407
substitution was predicted to be the cause of disease via multiple computational
408
predictive algorithms. To detect the effect of SOX3 c.424C>A; p.P142T on both
409
protein synthesis and function, we performed transient expression studies in HEK
410
293T and CHO cells with the WT and p.P142T mutant SOX3 cDNA constructs.
411
Western blotting and immunofluorescence staining showed no significant difference
412
between WT and mutant with regard to the expression level and subcellular
413
localization of SOX3. Luciferase assay showed increased transcriptional activation of
414
the SOX3 p.P142T mutation, indicating that the mutation may affect the binding
415
between SOX3 and the SOX3 target gene promoters and result in increased target
416
gene activation. The Wnt / β-catenin signaling pathway is important for pituitary
417
development and is required both within the pouch and in the ventral diencephalon
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(Kelberman et al., 2009; Olson et al., 2006). SOX3 has been shown to be capable of
419
repressing the β-catenin mediated activation (Alatzoglou et al., 2011; Wong et al.,
420
2007). To monitor the regulatory role of SOX3 p.P142T in modulating the
421
β-catenin-mediated transcription, TOPFLASH assay was performed. Although the
422
β-catenin mediated activation was suppressed by the SOX3 p.P142T mutant in a
423
dose-dependent fashion, the repression ability was attenuated compared to that of WT
424
SOX3. This indicates that the interplay between SOX3 and the Wnt / β-catenin
425
signaling pathway during pituitary organogenesis may be interrupted and those
426
β-catenin target genes may be inappropriately regulated. The precise mechanism by
427
which SOX3 regulates Wnt / β-catenin pathway is still being resolved. SOX3 has
428
been shown to repress the Wnt / β-catenin pathway by physically interacting with
429
β-catenin or binding to the Wnt-target gene promoters (Zhang et al., 2003; Zorn et al.,
430
1999). Whether p.P142T mutation attenuates the interaction between SOX3 and
431
β-catenin or affects the binding between SOX3 and the Wnt-target gene promoters
432
needs further study. Both in silico analysis and in vitro study suggest that SOX3
433
c.424C>A; p.P142T could be responsible for the X-linked hypopituitarism in this
434
family. However, the exact mechanism of SOX3 p.P142T in hypopituitarism and the
435
mechanisms underlying the observed phenotypic variability and GH independent
436
growth require further work.
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In conclusion, the novel SOX3 missense variant c.424C>A; p.P142T was identified in
439
an X-linked hypopituitarism pedigree of variable phenotypes. Despite severe GH
440
deficiency, the growth failure of the affected siblings was relatively modest. The
441
coexistent insulin insensitivity and compensatory hyperinsulinemia may contribute to
442
the maintained growth. The SOX3 variant was inherited from their unaffected mother.
443
In vitro study showed that this variant resulted in increased transcriptional activation
444
and impaired repression of β-catenin-mediated transcription, while both the
445
expression and nuclear targeting of SOX3 remained unaffected. Our study describes a
446
rare pathogenic point mutation in SOX3 and provides additional evidence in support
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of an association between SOX3 dysfunction and hypopituitarism.
448
449
Conflict of interest
450
The authors declare no conflict of interest.
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Acknowledgments
453
We thank all the members of the family for their participation in this study. This study
454
was support by Shanghai Pujiang Talent Program (Grant No.16PJ1406300), the
455
National Natural Science Foundation of China (Grant No. 8147205, 181772303), the
456
Project of Shanghai Municipal Science and Technology Commission (Grant No.
457
16ZR1421700, 15410722800, 17ZR1449100) and the Project of Shanghai Municipal
458
Education Commission-Gaofeng Clinical Medicine (Grant No. 20152529).
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Zhang,
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Figure legends
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Figure 1. The pedigree, growth curve, and molecular analysis of a Chinese family
573
showing X-linked hypopituitarism. (a) Family pedigree. (b) The Height of III3 and
574
III4 from 3 to 12.5 yrs. (c) Chromatogram of Sanger sequencing, showing
575
hemizygous and heterozygous SOX3 c.424C>A in III3 and the mother, and SOX3 WT
576
in the father. The corresponding sequences where the mutation was found are
577
indicated with arrows.
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Figure 2. Sagittal, coronal, and transverse cerebral MRI scans of III3 (a) and III4 (b),
580
showing anterior pituitary hypoplasia, pituitary stalk hypoplasia, and ectopic posterior
581
pituitary. Note that corpus callosum hypogenesis and midline lipoma were also
582
detected in III4. AP: anterior pituitary; PP: posterior pituitary; CC: corpus callosum; L:
583
lipoma.
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Figure 3. Conservation analysis and homology modeling of SOX3. (a) Alignment of
586
amino acid sequences of SOX3 in different species. (b) Alignment of amino acid
587
sequences among various human SOX transcription factors. The corresponding
588
residues of SOX3 P142 are shown in red. (c) Homology model of the wild type SOX3
589
DNA binding domain with an oligonucleotide. (d) Homology model of the p.P142T
590
mutant SOX3 DNA binding domain with an oligonucleotide. The SOX3 DNA binding
591
domain and the oligonucleotide are shown in green and orange, respectively, with the
592
SOX3 P142 residue shown in red.
593
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Figure 4. SOX3 p.P142T expression was comparable to that of the WT. (a) The
595
expression of SOX3 detected via western blot assay. Results are representative of at
596
least three independent experiments. (b) Quantification and statistical analysis of
597
SOX3 expression. Basically, the protein bands of SOX3 and β-actin were selected
598
respectively. The grey signal of each band was measured by ImageJ. The ratio of
599
SOX3 to the β-actin of the same sample was then calculated and the ratio of SOX3
600
WT to its β-actin was set as 100%.
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Figure 5. Subcellular localization analysis of the SOX3 via immunofluorescence
603
staining. Both WT and p.P142T mutant SOX3 are localized in the nucleus.
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Figure 6. (a) Transactivation assays of SOX3 using HESX1 promoter reporter. The
606
SOX3 p.P142T resulted in an up to 2.5-fold increase in transcriptional activation
607
compared to SOX3 WT. (b) TOPFLASH assay of SOX3. SOX3 p.P142T showed
608
significantly less repression activity compared to SOX3 WT. Values are expressed as
609
means ± standard deviation (n = 3).
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620
621
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623
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***
P < 0.001.
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Table 1 The results of GnRH stimulation test
Time (min)
LH (mUI/mL)
FSH (mUI/mL)
0
1.25
2.08
30
4.41
4.07
60
4.8
5.28
90
4.6
5.86
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630
631
Glucose (mmol/L)
Insulin (uIU/ml)
C-peptide (ng/ml)
0
5.1
12.3
2.23
7.3
101.6
9.7
4.7
17.4
4.52
5.9
57.7
7.38
60
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632
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Highlights
Identification of a novel SOX3 p.P142T variant associated with hypopituitarism
Modest growth failure in spite of the complete growth hormone deficiency
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Increased transcriptional activation of SOX3 p.P142T variant
Reduced ability of SOX3 p.P142T variant to repress β-catenin-mediated
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transcription
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