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A Case of Two Sisters Suffering from 46,XY Gonadal Dysgenesis and Carrying a Mutation of a Novel Candidate Sex-Determining Gene STARD8 on the X Chromosome

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Short Report

Sex Dev

DOI: 10.1159/000489692

A Case of Two Sisters Suffering from 46,XY

Gonadal Dysgenesis and Carrying a Mutation of a Novel Candidate Sex-Determining Gene STARD8 on the X Chromosome

Erkut Ilaslan

a

Pierre Calvel

d, e

Dominika Nowak

a

Maria Szarras-Czapnik

b

Jolanta Slowikowska-Hilczer

c

Anna Spik

a

Pauline Sararols

d

Serge Nef

d

Jadwiga Jaruzelska

a

Kamila Kusz-Zamelczyk

a

a Institute of Human Genetics, Polish Academy of Sciences, Poznan , b Department of Endocrinology and Diabetes, The Children’s Memorial Health Institute, Warsaw , and c Department of Andrology and Reproductive Endocrinology, Medical University of Lodz, Lodz , Poland; d Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, Geneva , Switzerland; e GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas , France

namely in testes determination and testosterone synthesis.

However, further studies are needed to confirm the involve- ment of STARD8 in sexual development.

© 2018 S. Karger AG, Basel

Development of male sex characteristics in 46,XY em- bryos depends on the expression of specific genes critical for testes determination as well as genes necessary for the synthesis and action of testicular hormones. Disruption of those genes may cause a spectrum of phenotypes of 46,XY disorders of sexual development (DSD). Despite a significant progress in understanding the mechanisms of male sexual development, the genetic background of

∼ 50% of 46,XY DSD cases remains to be uncovered [Eg- gers et al., 2016]. Therefore, identification of novel genes Keywords

Disorders of sexual development · STAR · STARD8 · Testosterone synthesis

Abstract

Identification of novel genes involved in sexual develop- ment is crucial for understanding disorders of sex develop- ment (DSD). Here, we propose a member of the START do- main family, the X chromosome STARD8, as a DSD candidate gene. We have identified a missense mutation of this gene in 2 sisters with 46,XY gonadal dysgenesis, inherited from their heterozygous mother. Gonadal tissue of one of the sis- ters contained Leydig cells overloaded with cholesterol droplets, i.e., structures previously identified in 46,XY DSD patients carrying mutations in the STAR gene encoding an- other START domain family member, which is crucial for ste- roidogenesis. Based on the phenotypes of our patients, we propose a dual role of STARD8 in sexual development,

Accepted: January 2, 2018 by M. Schmid

Published online: June 8, 2018

Kamila Kusz-Zamelczyk Institute of Human Genetics

Polish Academy of Sciences, Strzeszynska 32 PL–60-479 Poznan (Poland)

E-Mail kamila.kusz-zamelczyk   @   igcz.poznan.pl Serge Nef

© 2018 S. Karger AG, Basel

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involved in those pathways is of great importance to get a more complete insight into the genetic basis of human sex determination.

The STAR-related lipid-transfer (START) protein fam- ily encompasses a wide range of proteins characterized by the presence of the lipid-binding START domain. These proteins are responsible for proper lipid distribution in cells [Clark, 2012]. A founder member of this family, the steroidogenic acute regulatory protein (STAR), is known to be involved in male sex development since it plays a critical role in testosterone biosynthesis. STAR is expressed primarily in steroid-producing cells, including Leydig cells of the testes, theca, and luteal cells of the ovaries, as well as adrenal cortex cells. The START domain of STAR binds cholesterol and transfers it to the inner membrane of mi- tochondria, where the first enzymatic step of all steroid syntheses takes place [Clark et al., 1994; Sugawara et al., 1995]. STAR gene mutations lead to blockage of the syn- thesis of testosterone and other steroids, causing lipoid congenital adrenal hyperplasia, which in addition to adre- nal features is characterized by DSD in 46,XY individuals [Lin et al., 1995; Bose et al., 1996; Bhangoo et al., 2005]. At the cellular level, STAR mutations cause an abnormal ac- cumulation of cholesterol droplets within the cytoplasm of Leydig and adrenal cortex cells [Bose et al., 1996]. Beside the STAR protein, no other START family member has been described to be implicated in sexual development.

Here, we describe a mutation of the X chromosomal STARD8 gene (STAR-related lipid transfer domain con- taining 8) in 2 sisters suffering from 46,XY gonadal dys- genesis, which they inherited from their heterozygous mother. Based on the phenotypes of our patients, we pro- pose a dual role of STARD8 in sexual development, name- ly in testes determination and testosterone synthesis.

Case Report

Two sisters suffering from 46,XY DSD were analyzed in the present study.

Patient 1 was diagnosed at the age of 17 years because of a lack of signs of puberty and menstruation. She was characterized by female-type external genitalia, oviducts, a normal prepubertal va- gina, and uterus in spite of a 46,XY karyotype. Small gonads lo- cated at the normal ovarian position were detected by ultrasonog- raphy. Hormonal analysis revealed a very low level of testosterone and estradiol, despite a high level of gonadotropins, which is typi- cal for hypergonadotropic hypogonadism. The patient was sub- jected to bilateral gonadectomy.

Patient 2 was diagnosed at the age of 8 months because of am- biguous external genitalia (labioscrotum, perineal urethral orifice and vaginal opening, phallic tubercle ca. 150 mm long) on the background of a 46,XY karyotype. The patient had oviducts, nor-

mal prepubertal vagina, and uterus. Both gonads were located in the ovarian position. Gonadotropin and testosterone serum levels were low, typical for the prepubertal period, and a hCG test re- vealed impaired synthesis of testosterone. The child was subjected to bilateral gonadectomy.

Methods

Array Comparative Genome Hybridization

Genomic DNA samples from the probands, their parents, and their healthy brother were isolated from blood samples by using the DNA mini kit (Qiagen, Hilden, Germany) and were tested us- ing the Agilent SurePrint G3 CGH Microarray Kit, 1x1M (Agilent Technologies, Santa Clara, CA, USA). Labeling and hybridization were performed according to the manufacturer’s protocol. Data were analyzed using Agilent Genomic Workbench, and probe po- sitions were determined according to NCBI37/hg19 build.

Whole Exome Sequencing

Exome capture was performed on DNA from the patients, their parents, and a healthy brother by using the SureSelect Human All Exon v3 kit (Agilent Technologies). Sequencing was performed on an Illumina HiSeq 2000 device, and Fastq files were obtained using the Illumina CASAVA v1.8.1 software. The raw data were analyzed using our bioinformatic pipeline hosted on the Vital-IT Center of the Swiss Institute of Bioinformatics (SIB; http://www.vital-it.ch) as described previously [Callier et al., 2014]. Whole exome se- quencing (WES) data were analyzed using the VariantMaster soft- ware [Santoni et al., 2014] in order to identify de novo variants as well as variants with different Mendelian inheritance models (dominant with reduced penetrance, recessive, and X-linked). The mutation identified in the STARD8 gene was validated by Sanger sequencing in the patients and compared to their parents and their healthy brother.

Histochemistry

Histological characteristics were examined in paraffin-embed- ded sections of gonadal tissue specimens. The general structure of the gonadal tissues was assessed by standard hematoxylin-eosin staining.

Accession Numbers

Accession numbers of Homo sapiens STARD8 used are NM_014725 and NP_055540.

Results

Identification of the p.Ser913Asn STARD8 Mutation While searching for the genetic background of 46,XY DSD in our patients, no mutations were identified in the SRY gene (not shown). Therefore, we performed WES to identify some single nucleotide variants (SNVs) underly- ing the disease, as well as array comparative genomic hy- bridization (aCGH) to detect potential causative copy number variations. aCGH revealed only several copy

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number variations known as nonpathogenic. Among the 22,645 and the 22,675 SNVs identified by WES in patient 1 and patient 2, respectively (online suppl. Table 1; see www.karger.com/doi/10.1159/000489692 for all online suppl. material), we focused on the p.Ser913Asn substitu- tion caused by a c.2738G>A transition in the STARD8 gene on the X chromosome, which was detected in both patients. Importantly, this mutation was also present in the heterozygous mother but absent in the father as well as in the healthy brother ( Fig. 1 ). Moreover, this mutation was located within the STAR functional domain. Notably, the position of the p.Ser913Asn substitution is conserved in vertebrates (online suppl. Fig. 1). No mutations in oth- er genes supposed to play a role in DSD were identified (online suppl. Table 2).

Histological Phenotypes of Gonads of the Patients Since the STARD8 gene belongs to the same family as the STAR gene, mutations of which were previously de- scribed as causing 46,XY DSD, we sought to perform his- topathological analyses of our patients to compare them with histological phenotypes of the previously reported pa- tients carrying STAR gene mutations [Khoury et al., 2016].

A histological analysis of gonads of patient 1 revealed dysgenetic gonads composed of ovarian-like connective

a b

c d

Fig. 1. Identification of the STARD8 p.Se- r913Asn mutation in patients 1 and 2. The pedigree shows the inheritance of the mu- tation according to the X-linked recessive model. The results of DNA sequencing show the c.2738G>A substitution status for each family member. Black circles repre- sent the affected sisters carrying the hemi- zygous mutation, a gray circle with a black dot represents the unaffected mother car- rying the heterozygous mutation, whereas gray squares represent the unaffected brother and father, carrying the hemizy- gous wild-type allele. The location of the p.Ser913Asn mutation in the START do- main of the STARD8 protein is shown at the bottom.

Fig. 2. Histological analysis of the patients’ gonads stained with hematoxylin-eosin. a Numerous clarified Leydig-like cells (Ll) reminiscent of cells overloaded with cholesterol droplets are visible below the ovarian-like stroma (Os) in patient 1. White rectangle encompasses Leydig-like cells. Scale bar, 20 μm. b Magnified im- age of clarified Leydig-like cells from a . Scale bar, 10 μm. c Semi- niferous tubules containing immature Sertoli cells (SC), gonocytes (G), spermatogonia (Sg), while fibroblast-like cells (Fl) are present in the intertubular space in patient 2. Scale bar, 20 μm. d Fibro- blast-like cells (Fl) and single seminiferous tubules containing im- mature Sertoli cells (SC) and gonocytes (G). Scale bar, 20 μm.

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tissue, but lacking the ovary or testis structures. Below the gonadal cortex, large aggregations of Leydig-like cells were present, and a significant number of them had a clarified cytoplasm characteristic of cholesterol accumu- lation ( Fig. 2 a, b). Finally, 46,XY complete gonadal dys- genesis was diagnosed in this patient.

A histological analysis of the gonads of patient 2 re- vealed that the right gonad was a streak of connective tis- sue, lacking testicular or ovarian structures but contain- ing numerous fibroblast-like cells. The left gonad instead had a partly testicular structure. There were 2 types of adjacent tissues: (a) testicular tissue similar to the normal equivalent (considering the age of the patient), composed of tubules with slightly larger diameter (95–125 μm, mean 113.7 μm) and slightly thicker tubular membranes (3.1–

5.6 μm, mean 3.7 μm), containing immature Sertoli cells, fetal germ cells (gonocytes), single spermatogonia, and intertubular space containing fibroblast-like cells ( Fig. 2 c), and (b) an abnormal testicular tissue composed of fibroblast-like cells and rare seminiferous tubules of smaller diameter (43.1–92.3 μm, mean 76.5 μm), contain- ing immature Sertoli cells and single gonocytes ( Fig. 2 d).

Finally, 46,XY asymmetric gonadal dysgenesis was diag- nosed in this patient.

Discussion

In this work, we describe the p.Ser913Asn STARD8 gene mutation in 2 sisters manifesting with 46,XY gonad- al dysgenesis. The role of STARD8 is not established yet, since no mutations of this gene related to any pathological phenotype have been described so far. STARD8 belongs to the START domain protein family, which contains a conserved lipid-binding START domain responsible for proper lipid distribution in cells [Clark, 2012]. Impor- tantly, the p.Ser913Asn amino acid substitution in STARD8 is located within this domain and, notably, at a position that is conserved among vertebrates (online suppl. Fig. 1). This may suggest a causative effect of this serine amino acid substitution for the STARD8 dysfunc- tion. Moreover, the p.Ser913Asn STARD8 substitution is a very rare variant (rs201005000) in populations world- wide, the highest minor allele frequency being <0.01 [McLaren et al., 2016].

The presence of dysgenetic gonad(s) in both 46,XY pa- tients carrying the STARD8 mutation strongly suggests a role of this gene in testicular determination. The male sex-specific Stard8 expression in developing murine go- nads during the time critical for sex determination in

mice is an argument supporting STARD8 function in go- nadal sex determination [Nef et al., 2005; Jameson et al., 2012].

The STARD8 mutation occurred within the START domain. Interestingly, mutations within the START do- main of the STAR protein (which is another member of the START family) cause 46,XY DSD, coexisting with li- poid congenital adrenal hyperplasia [Lin et al., 1995; Bose et al., 1996; Bhangoo et al., 2005]. This phenotype is due to a deficient steroidogenesis in gonads and adrenal glands, as STAR controls a crucial step for steroid biosyn- thesis, namely the transfer of cholesterol from the outer to the inner mitochondrial membrane. The blockage of cholesterol transport due to STAR gene mutations causes cholesterol accumulation in the cytoplasm in the form of droplets [Sugawara et al., 1995; Bose et al., 1996]. This cholesterol overloading of Leydig cells is manifested by clarified cytoplasm [Khoury et al., 2016]. It is noteworthy that we observed such Leydig cells with clarified cyto- plasm in patient 1 ( Fig. 2 a, b). This abnormal phenotype of Leydig cells, suggesting testosterone synthesis block- age, is in line with the low testosterone level in patient 1, which was measured before gonadectomy. However, in the gonads of patient 2 we did not observe Leydig cells overloaded with cholesterol, probably because of her young age (8 months) when gonadectomy was performed, as gonads are not hormonally active in this period of life.

Nevertheless, coexistence of 3 features – the STARD8 mu- tation, the Leydig cell phenotype characteristic for tes- tosterone synthesis blockage in patient 1, and the low testosterone levels in patient 1 (in pubertal age) and pa- tient 2 (in infancy, as measured by the hCG test) – may suggest a causative nature of the p.Ser913Asn STARD8 mutation described here as well as the role of STARD8 in gonadal steroidogenesis, possibly synergistic to STAR. In addition, its similar cytoplasmic/membranous location within steroidogenic Leydig cells of adult males supports a synergistic role of those 2 proteins in testosterone bio- synthesis [Uhlen et al., 2015]. Interestingly, in contrast to patients carrying STAR mutations, our patients had no features of adrenal failure. Since our report is the first one describing a STARD8 mutation in 46,XY DSD patients, we cannot exclude that there are other types of STARD8 mutations associated also with adrenal insufficiency.

Such only partially overlapping phenotypes could exist, like in patients carrying mutations of another sex deter- mination gene, NR5A1 . In some patients, NR5A1 gene mutations caused 46,XY DSD with adrenal failure, while in other 46,XY DSD patients, various anomalies of testis development were identified, but with no evidence of ad-

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renal failure. The latter group includes a majority of cases carrying NR5A1 mutations [Domenice et al., 2016].

Altogether, the coexistence of the STARD8 p.Ser913Asn hemizygous mutation in association with 46,XY DSD in 2 patients, the location of the mutation at a conserved po- sition within the functional START domain, gonadal dys- genesis in both patients, and sex-specific expression of Stard8 in differentiating gonads of mice suggest that the STARD8 gene may play a role in male determination of primary gonads. On the other hand, the presence of large numbers of Leydig cells or Leydig cell precursors in com- bination with low testosterone levels and the pathological phenotype of Leydig cells indicating cholesterol accumu- lation suggest a role of STARD8 in testosterone synthesis.

Identification of other 46,XY DSD patients with STARD8 mutations is necessary to confirm the significance of this gene in sexual development.

Acknowledgments

This study was supported by the Swiss National Science Foun- dation (joint research project SCOPES IZ73Z0_152347/1 to S.N.

and J.J.).

Statement of Ethics

All clinical investigations were performed according to the Declaration of Helsinki principles. The study was approved by the Bioethics Committee at Poznan University of Medical Sciences (authorization number 817/13) and the Genova Ethical Commit- tee (CCER, authorization number 14-121).

Disclosure Statement

The authors have no conflicts of interest to declare.

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