Publications by authors named "Remko Hersmus"

34 Publications

Under-reported aspects of diagnosis and treatment addressed in the Dutch-Flemish guideline for comprehensive diagnostics in disorders/differences of sex development.

J Med Genet 2020 09 17;57(9):581-589. Epub 2020 Apr 17.

Department of Pediatric Endocrinology and DSD Expert Center ErasmusMC, Erasmus Medical Center, 3015 GD Rotterdam, The Netherlands.

We present key points from the updated Dutch-Flemish guideline on comprehensive diagnostics in disorders/differences of sex development (DSD) that have not been widely addressed in the current (inter)national literature. These points are of interest to physicians working in DSD (expert) centres and to professionals who come across persons with a DSD but have no (or limited) experience in this area. The Dutch-Flemish guideline is based on internationally accepted principles. Recent initiatives striving for uniform high-quality care across Europe, and beyond, such as the completed COST action 1303 and the European Reference Network for rare endocrine conditions (EndoERN), have generated several excellent papers covering nearly all aspects of DSD. The Dutch-Flemish guideline follows these international consensus papers and covers a number of other topics relevant to daily practice. For instance, although next-generation sequencing (NGS)-based molecular diagnostics are becoming the gold standard for genetic evaluation, it can be difficult to prove variant causality or relate the genotype to the clinical presentation. Network formation and centralisation are essential to promote functional studies that assess the effects of genetic variants and to the correct histological assessment of gonadal material from DSD patients, as well as allowing for maximisation of expertise and possible cost reductions. The Dutch-Flemish guidelines uniquely address three aspects of DSD. First, we propose an algorithm for counselling and diagnostic evaluation when a DSD is suspected prenatally, a clinical situation that is becoming more common. Referral to ultrasound sonographers and obstetricians who are part of a DSD team is increasingly important here. Second, we pay special attention to healthcare professionals not working within a DSD centre as they are often the first to diagnose or suspect a DSD, but are not regularly exposed to DSDs and may have limited experience. Their thoughtful communication to patients, carers and colleagues, and the accessibility of protocols for first-line management and efficient referral are essential. Careful communication in the prenatal to neonatal period and the adolescent to adult transition are equally important and relatively under-reported in the literature. Third, we discuss the timing of (NGS-based) molecular diagnostics in the initial workup of new patients and in people with a diagnosis made solely on clinical grounds or those who had earlier genetic testing that is not compatible with current state-of-the-art diagnostics.
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http://dx.doi.org/10.1136/jmedgenet-2019-106354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476274PMC
September 2020

NR5A1 gene variants repress the ovarian-specific WNT signaling pathway in 46,XX disorders of sex development patients.

Hum Mutat 2019 02 30;40(2):207-216. Epub 2018 Nov 30.

Murdoch Children's Research Institute, Melbourne, Australia.

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.
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http://dx.doi.org/10.1002/humu.23672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492147PMC
February 2019

A Rare Case of Embryonal Carcinoma in a Patient with Turner Syndrome without Y Chromosomal Material but Mutations in KIT, AKT1, and ZNF358 Demonstrated Using Exome Sequencing.

Sex Dev 2017 2;11(5-6):262-268. Epub 2017 Dec 2.

Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark.

Gonadoblastoma and malignant transformations thereof can occur in females with Turner syndrome (TS) and Y chromosomal material. However, in females with TS and no Y chromosomal material, this is rarely seen. We report a female with an apparent 45,X karyotype (in blood and tumor) who was diagnosed with a metastatic embryonal carcinoma. Exome sequencing of blood and the tumor was done, and no Y chromosomal material was detected, while predicted deleterious mutations in KIT (likely driver), AKT1, and ZNF358 were identified in the tumor. The patient was treated with chemotherapy (first-line: cisplatin, etoposide, and bleomycin; second-line: paclitaxel and gemcitabine), and after that surgical debulking was performed. She is currently well and without signs of relapse. We conclude that embryonal carcinoma can apparently occur in 45,X TS without signs of Y chromosomal material.
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http://dx.doi.org/10.1159/000484398DOI Listing
October 2018

Histological Assessment of Gonads in DSD: Relevance for Clinical Management.

Sex Dev 2018 3;12(1-3):106-122. Epub 2017 Nov 3.

Developmental Endocrinology Research Group, School of Medicine, University of Glasgow, Glasgow, UK.

Malignant gonadal germ cell tumors, referred to as germ cell cancers (GCC), occur with increased frequency in individuals who have specific types of differences (disorders) of sex development (DSD). Recent population-based studies have identified new environmental and genetic risk factors that have led to a 'genvironment' hypothesis, which may potentially be helpful in risk assessment in DSD-related GCC. In DSD, the malignancy risk is highly heterogeneous, but recent studies allow now to discriminate between high- and low-risk conditions. Gonadal biopsy is in some cases the best procedure of choice to assess the risk, and with the availability of immunohistochemical biomarkers [OCT3/4 (POU5F1), TSPY, SOX9, FOXL2 and KITLG (SCF)], a reliable classification of GCC and its precursors can be made. The opportunities in the field of virtual diagnostic pathology will be presented, having possibilities for rare diseases in general and DSD specifically. It is expected that the International DSD Registry will stimulate international collaborations, facilitating better diagnostic procedures as well as research.
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http://dx.doi.org/10.1159/000481757DOI Listing
November 2018

Functional characterization of novel NR5A1 variants reveals multiple complex roles in disorders of sex development.

Hum Mutat 2018 01 2;39(1):124-139. Epub 2017 Nov 2.

Murdoch Children's Research Institute, Melbourne, Australia.

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.
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http://dx.doi.org/10.1002/humu.23354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765430PMC
January 2018

Multiparameter Investigation of a 46,XX/46,XY Tetragametic Chimeric Phenotypical Male Patient with Bilateral Scrotal Ovotestes and Ovulatory Activity.

Sex Dev 2018 20;12(1-3):145-154. Epub 2017 Sep 20.

Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands.

We report on an adult male initially presenting with gynecomastia and a painless scrotal mass without additional genital anomalies. Hyperpigmentation of the skin following the Blaschko's lines was identified. He underwent gonadectomy because of suspected cancer. Histological analyses revealed an ovotestis with ovulatory activity confirmed by immunohistochemistry with multiple markers. Karyotyping of cultured peripheral blood lymphocytes and a buccal smear revealed a 46,XX/46,XY chimeric constitution with different percentages. Multiple molecular analyses as well as blood typing implied a tetragametic origin. After the unilateral gonadectomy, the patient developed recurrent painful cystic swellings of the remaining gonad. Because of the wish to preserve hormonal activity as well as future fertility, the patient underwent surgical resection of a cystic gonadal area. The removed tissue showed ovulation-related features in addition to both testicular and ovarian tissue, diagnosed as an ovotestis. Testosterone therapy was initiated to suppress the persistently elevated gonadotropins and thereby suppress ovarian activity. During treatment, the recurrent pain complaints and cystic swellings ceased, although gonadotropin levels were not fully suppressed. Based on these observations, the importance of a detailed genetic and pathological diagnosis and the clinical dilemmas including the pros and cons of personalized treatment with gonadal preservative surgery are discussed.
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http://dx.doi.org/10.1159/000479946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872565PMC
November 2018

The biology of germ cell tumors in disorders of sex development.

Clin Genet 2017 02 24;91(2):292-301. Epub 2016 Nov 24.

Department of Pathology, Erasmus University Medical Center, Rotterdam, The Netherlands.

Development of a malignant germ cell tumor, i.e., germ cell cancer (GCC) in individuals with disorders of sex development (DSD) depends on a number of (epi-)genetic factors related to early gonadal- and germ cell development, possibly related to genetic susceptibility. Fetal development of germ cells is orchestrated by strict processes involving specification, migration and the development of a proper gonadal niche. In this review we will discuss the early (epi-)genetic events in normal and aberrant germ cell and gonadal development. Focus will be on the formation of the precursor lesions of GCC in individuals who have DSD. In our view, expression of the different embryonic markers in, and epigenetic profile of the precursor lesions reflects the developmental stage in which these cells are blocked in their maturation. Therefore, these are not a primary pathogenetic driving force. Progression later in life towards a full blown cancer likely depends on additional factors such as a changed endocrine environment in a susceptible individual. Genetic susceptibility is, as evidenced by the presence of specific risk genetic variants (SNPs) in patients with a testicular GCC, related to genes involved in early germ cell and gonadal development.
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http://dx.doi.org/10.1111/cge.12882DOI Listing
February 2017

Hormonal evaluation in relation to phenotype and genotype in 286 patients with a disorder of sex development from Indonesia.

Clin Endocrinol (Oxf) 2016 08 4;85(2):247-57. Epub 2016 Apr 4.

Department of Paediatrics, Division of Endocrinology, Sophia Children's Hospital, Erasmus University Medical Centre, Rotterdam, The Netherlands.

Objective: The objective of this study was to determine the aetiological spectrum of disorders of sex development (DSD) in a large cohort of underprivileged and undiagnosed patients from Indonesia.

Methods: A total of 286 patients with atypical external and/or internal genitalia were evaluated using clinical, hormonal, molecular genetic and histological parameters.

Results: The age (years) at presentation was 0-0·5 in 41 (14·3%), >0·5-12 in 181 (63·3%) and >12 in 64 cases (22·4%). 46,XY DSD was most common (68·2%, n = 195), 46,XX DSD was found in 23·4% (n = 67) and sex chromosomal DSD in 8·4% (n = 24). In 61·2% of 46,XX DSD patients, 17·9% of 46,XY DSD patients and all sex chromosome DSD patients (29·4% in total), a final diagnosis was reached based on genetic or histological gonadal tissue evaluation. 17-hydroxyprogesterone and androstenedione levels were the most distinctive parameters in 46,XX DSD patients. In 46,XY DSD, diagnostic groups were identified based on the external masculinization score: androgen action disorder (AAD), unknown male undermasculinization (UMU), and gonadal dysgenesis (GD). LH, FSH and testosterone levels were most informative especially in the older age group. HCG tests were of no additional value as no patients with androgen synthesis disorders were found. Hormonal profiles of patients with sex chromosome DSD and a Y-chromosome sequence containing karyotype showed high levels of LH and FSH, and low levels of AMH, inhibin B and testosterone compared with the normal male range. Gene mutations were found in all patients with CAH, but in only 24·5% and 1·8% of patients with AAD and UMU. In 32% of 46,XY GD patients, copy number variants of different genes were found.

Conclusion: A stepwise diagnostic approach led to a molecularly or histologically proven final diagnosis in 29·4% of the patients. The most informative parameters were serum levels of 17-hydroxyprogesterone and androstenedione in 46,XX DSD patients, and serum LH, FSH and testosterone levels in 46,XY DSD patients.
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http://dx.doi.org/10.1111/cen.13051DOI Listing
August 2016

Aberrant SOX2 expression in colorectal cancers does not correlate with mucinous differentiation and gastric mucin MUC5AC expression.

Virchows Arch 2014 Oct 10;465(4):395-400. Epub 2014 Aug 10.

Department of Pediatric Surgery, Erasmus Medical Centre, Rotterdam, The Netherlands.

Colorectal cancer (CRC) can be divided into non-mucinous and mucinous subtypes, of which the latter portends to have a worse clinical prognosis. A previous study suggested a putative link between SOX2 expression observed selectively in mucinous CRC and the induction of the gastric mucin MUC5AC. In this study, we re-evaluated the expression behavior of SOX2, MUC5AC, and CDX2 in both types of CRC. We performed immunohistochemical analysis on 90 cases of non-mucinous CRCs, 57 cases of mucinous CRCs, and 15 case-matched normal intestinal mucosa. In contrast to the previously suggested link between SOX2 and mucinous CRC, we observe aberrant expression of SOX2 at equal levels in both subtypes. Fluorescence in situ hybridization (FISH) analysis shows that expression is not attributed to genomic amplification. While SOX2 and CDX2 are normally expressed in a reciprocal manner, SOX2-positive tumor cells co-express CDX2. Furthermore, we show that MUC5AC is expressed independently of SOX2. In conclusion, we show that aberrant SOX2 expression is specifically linked neither to mucinous CRCs nor to the induction of MUC5AC, in contrast to previous suggestions.
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http://dx.doi.org/10.1007/s00428-014-1638-yDOI Listing
October 2014

Gonadal malignancy in 13 consecutive collected patients with disorders of sex development (DSD) from Semarang (Indonesia).

J Clin Pathol 2013 Mar 25;66(3):198-204. Epub 2012 Dec 25.

Division of Human Genetics Center for Biomedical Research, Faculty of Medicine Diponegoro University (FMDU), Semarang, Central Java, Indonesia.

Aims: Caucasian patients with disorders of sex development (DSD) are at a high risk of developing germ cell cancer (GCC). GCC is prominent in young adults in Western countries, while the incidence is significantly lower in Asia. So far, the risk of GCC in Asian DSD patients is unknown.

Methods And Results: A detailed study of gonad histology , morphology and immunohistochemistry (OCT3/4, testis-specific protein Y-encoded, VASA, SCF/KITLG, SOX9, FOXL2) of 16 Indonesian DSD patients was undertaken. 13 cases could be analysed, including ovarian tissue (n=3), streak gonad (n=1), undifferentiated gonad (n=1) and testicular tissue (n=8), diagnosed as 46, XX (n=1), 46, XY (n=7) and sex chromosome DSD (n=5). The precursor lesion gonadoblastoma or carcinoma in situ, or GCC was diagnosed in four cases (30.8%; three 46, XY and one sex chromosome DSD ). A hormone producing ovarian Leydig cell tumour was identified in a 46, XX patient, supposed to be a late onset congenital adrenal hyperplasia.

Conclusions: In spite of the significantly lower risk of GCC in the general Asian population, DSD is a dominant risk factor. The study demonstrates the power of immunohistochemical markers for (early) diagnosis. This knowledge will deepen understanding of the pathobiology of GCC and clinical handling of patients with DSD, globally.
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http://dx.doi.org/10.1136/jclinpath-2012-201062DOI Listing
March 2013

SRY mutation analysis by next generation (deep) sequencing in a cohort of chromosomal Disorders of Sex Development (DSD) patients with a mosaic karyotype.

BMC Med Genet 2012 Nov 16;13:108. Epub 2012 Nov 16.

Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Josephine Nefkens Institute, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Background: The presence of the Y-chromosome or Y chromosome-derived material is seen in 4-60% of Turner syndrome patients (Chromosomal Disorders of Sex Development (DSD)). DSD patients with specific Y-chromosomal material in their karyotype, the GonadoBlastoma on the Y-chromosome (GBY) region, have an increased risk of developing type II germ cell tumors/cancer (GCC), most likely related to TSPY. The Sex determining Region on the Y gene (SRY) is located on the short arm of the Y-chromosome and is the crucial switch that initiates testis determination and subsequent male development. Mutations in this gene are responsible for sex reversal in approximately 10-15% of 46,XY pure gonadal dysgenesis (46,XY DSD) cases. The majority of the mutations described are located in the central HMG domain, which is involved in the binding and bending of the DNA and harbors two nuclear localization signals. SRY mutations have also been found in a small number of patients with a 45,X/46,XY karyotype and might play a role in the maldevelopment of the gonads.

Methods: To thoroughly investigate the presence of possible SRY gene mutations in mosaic DSD patients, we performed next generation (deep) sequencing on the genomic DNA of fourteen independent patients (twelve 45,X/46,XY, one 45,X/46,XX/46,XY, and one 46,XX/46,XY).

Results And Conclusions: The results demonstrate that aberrations in SRY are rare in mosaic DSD patients and therefore do not play a significant role in the etiology of the disease.
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http://dx.doi.org/10.1186/1471-2350-13-108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538515PMC
November 2012

Prevalence of c-KIT mutations in gonadoblastoma and dysgerminomas of patients with disorders of sex development (DSD) and ovarian dysgerminomas.

PLoS One 2012 28;7(8):e43952. Epub 2012 Aug 28.

Department of Pathology, Erasmus MC - University Medical Center Rotterdam, Josephine Nefkens Institute, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Activating c-KIT mutations (exons 11 and 17) are found in 10-40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ~3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043952PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429439PMC
February 2013

A 46,XY female DSD patient with bilateral gonadoblastoma, a novel SRY missense mutation combined with a WT1 KTS splice-site mutation.

PLoS One 2012 18;7(7):e40858. Epub 2012 Jul 18.

Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Josephine Nefkens Institute, Rotterdam, The Netherlands.

Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10-15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms' tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice-site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040858PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3399878PMC
March 2013

Delayed Recognition of Disorders of Sex Development (DSD): A Missed Opportunity for Early Diagnosis of Malignant Germ Cell Tumors.

Int J Endocrinol 2012 19;2012:671209. Epub 2012 Jan 19.

Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Josephine Nefkens Institute, Daniel den Hoed Cancer Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Disorders of sex development (DSD) are defined as a congenital condition in which development of chromosomal, gonadal or anatomical sex is atypical. DSD patients with gonadal dysgenesis or hypovirilization, containing part of the Y chromosome (GBY), have an increased risk for malignant type II germ cell tumors (GCTs: seminomas and nonseminomas). DSD may be diagnosed in newborns (e.g., ambiguous genitalia), or later in life, even at or after puberty. Here we describe three independent male patients with a GCT; two were retrospectively recognized as DSD, based on the histological identification of both carcinoma in situ and gonadoblastoma in a single gonad as the cancer precursor. Hypospadias and cryptorchidism in their history are consistent with this conclusion. The power of recognition of these parameters is demonstrated by the third patient, in which the precursor lesion was diagnosed before progression to invasiveness. Early recognition based on these clinical parameters could have prevented development of (metastatic) cancer, to be treated by systemic therapy. All three patients showed a normal male 46,XY karyotype, without obvious genetic rearrangements by high-resolution whole-genome copy number analysis. These cases demonstrate overlap between DSD and the so-called testicular dysgenesis syndrome (TDS), of significant relevance for identification of individuals at increased risk for development of a malignant GCT.
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http://dx.doi.org/10.1155/2012/671209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272341PMC
August 2012

Application of the new classification on patients with a disorder of sex development in indonesia.

Int J Endocrinol 2012 29;2012:237084. Epub 2011 Dec 29.

Department of Human Genetics, Center for Biomedical Research, Faculty of Medicine Diponegoro University (FMDU), Semarang 50321, Indonesia.

Disorder of sex development (DSD) patients in Indonesia most often do not receive a proper diagnostic evaluation and treatment. This study intended to categorize 88 Indonesian patients in accordance with the new consensus DSD algorithm. Diagnostic evaluation including clinical, hormonal, genetic, imaging, surgical, and histological parameters was performed. Fifty-three patients were raised as males, and 34 as females. Of 22 patients with 46, XX DSD, 15 had congenital adrenal hyperplasia, while in one patient, an ovarian Leydig cell tumor was found. In all 58 46, XY DSD patients, 29 were suspected of a disorder of androgen action (12 with an androgen receptor mutation), and in 9, gonadal dysgenesis was found and, in 20, severe hypospadias e.c.i. Implementation of the current consensus statement in a resource-poor environment is very difficult. The aim of the diagnostic workup in developing countries should be to end up with an evidence-based diagnosis. This is essential to improve treatment and thereby to improve the patients' quality of life.
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http://dx.doi.org/10.1155/2012/237084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255103PMC
August 2012

A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development.

Eur J Hum Genet 2012 Mar 9;20(3):348-51. Epub 2011 Nov 9.

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development.
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http://dx.doi.org/10.1038/ejhg.2011.204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3283189PMC
March 2012

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

J Mol Cell Biol 2010 Dec 6;2(6):333-44. Epub 2010 Oct 6.

College of Life Sciences, Nankai University, Tianjin 300071, China.

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.
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http://dx.doi.org/10.1093/jmcb/mjq029DOI Listing
December 2010

Gonadal tumours and DSD.

Best Pract Res Clin Endocrinol Metab 2010 Apr;24(2):291-310

Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Josephine Nefkens Institute, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Disorders of sex development (DSD), previously referred to as intersex, has been recognised as one of the main risk factors for development of type II germ cell tumours (GCTs), that is, seminomas/dysgerminomas and non-seminomas (e.g., embryonal carcinoma, yolk sac tumour, choriocarcinoma and teratoma). Within the testis, this type of cancer is the most frequent malignancy in adolescent and young adult Caucasian males. Although these males are not known to have dysgenetic gonads, the similarities in the resulting tumours suggest a common aetiological mechanism(s),--genetically, environmentally or a combination of both. Within the group of DSD patients, being in fact congenital conditions, the risk of malignant transformation of germ cells is highly heterogeneous, depending on a number of parameters, some of which have only recently been identified. Understanding of these recent insights will stimulate further research, with the final aim to develop an informative clinical decision tree for DSD patients, which includes optimal (early) diagnosis without overtreatment, such as prophylactic gonadectomy in the case of a low tumour risk.
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http://dx.doi.org/10.1016/j.beem.2009.10.002DOI Listing
April 2010

Systematic structure-function analysis of androgen receptor Leu701 mutants explains the properties of the prostate cancer mutant L701H.

J Biol Chem 2010 Feb 10;285(7):5097-105. Epub 2009 Dec 10.

Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands.

One mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). We investigated the unique properties of the AR L701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function analysis. Most amino acid substitutions at position 701 did not affect AR activation by 5alpha-dihydrotestosterone. Further analysis of the AR Leu(701) variants showed that AR L701M and AR L701Q, like AR L701H, had changed ligand responsiveness. AR L701M was strongly activated by progesterone but not by cortisol, whereas the opposite was observed for AR L701Q and AR L701H. Next, we analyzed a panel of structurally related steroids to study which of the OH groups at positions 11beta, 17alpha, and 21, which discriminate cortisol from progesterone, underlie the differential responses to both hormones. The results showed that the 17alpha-OH group was essential for activation of AR L701H and AR L701Q, whereas its absence was important for activation of AR L701M. Modeling indicated a conserved H-bonding network involving the steroidal 17alpha-OH group, His(701) or Gln(701), and the backbone of Ser(778). This network is absent in Leu(701) and in other mutants. A hydrophobic leucine or methionine at position 701 is unfavorable for the 17alpha-OH group. Our results indicate that the specific amino acid residue at position 701, its interaction with the backbone of Ser(778), and the steroidal 17alpha-hydroxyl group of the ligand are all important for the distinct transcriptional responses to progesterone and cortisol of AR mutants, including the prostate cancer mutant L701H.
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http://dx.doi.org/10.1074/jbc.M109.039958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836112PMC
February 2010

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma.

Eur J Hum Genet 2009 Dec 10;17(12):1642-9. Epub 2009 Jun 10.

Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Josephine Nefkens Institute, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10-15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age.
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http://dx.doi.org/10.1038/ejhg.2009.96DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2987026PMC
December 2009

Recent developments in testicular germ cell tumor research.

Birth Defects Res C Embryo Today 2009 Mar;87(1):96-113

Department of Pathology, Erasmus MC, Erasmus University Medical Center, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Rotterdam, The Netherlands.

Testicular germ cell tumors of adolescents and adults (TGCTs; the so-called type II variant) are the most frequent malignancies found in Caucasian males between 20 and 40 years of age. The incidence has increased over the last decades. TGCTs are divided into seminomas and nonseminomas, the latter consisting of the subgroups embryonal carcinoma, yolk-sac tumor, teratoma, and choriocarcinoma. The pathogenesis starts in utero, involving primordial germ cells/gonocytes that are blocked in their differentiation, and develops via the precursor lesion carcinoma in situ toward invasiveness. TGCTs are totipotent and can be considered as stem cell tumors. The developmental capacity of their cell of origin, the primordial germ cells/gonocyte, is demonstrated by the different tumor histologies of the invasive TGCTs. Seminoma represents the germ cell lineage, and embryonal carcinoma is the undifferentiated component, being the stem cell population of the nonseminomas. Somatic differentiation is seen in the teratomas (all lineages), whereas yolk-sac tumors and choriocarcinoma represent extra-embryonal differentiation. Seminomas are highly sensitive to irradiation and (DNA damaging) chemotherapy, whereas most nonseminomatous elements are less susceptible to radiation, although still sensitive to chemotherapy, with the exception of teratoma. To allow early diagnosis and follow up, appropriate markers are mandatory to discriminate between the different subgroups. In this review, a summary will be given related to several recent developments in TGCT research, especially selected because of their putative clinical impact.
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http://dx.doi.org/10.1002/bdrc.20140DOI Listing
March 2009

New insights into type II germ cell tumor pathogenesis based on studies of patients with various forms of disorders of sex development (DSD).

Mol Cell Endocrinol 2008 Sep 4;291(1-2):1-10. Epub 2008 Mar 4.

Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Rotterdam, The Netherlands.

Disorders of sex development (DSD), previously known as intersex, refer to congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. Patients with specific variants of this disorder have an elevated risk for the development of so-called type II germ cell cancers, i.e., the seminomatous and nonseminatous tumors, referred to as germ cell tumors (GCTs). Specifically DSD patients with gonadal dysgenesis or hypovirilization are at risk. A prerequisite for type II GCT formation is the presence of a specific part of the Y chromosome (referred to as the GBY region), with the TSPY gene being the most likely candidate. Also the octamer binding transcription factor OCT3/4 is consistently expressed in all type II GCTs with pluripotent potential, as well as in the precursor lesions carcinoma in situ (CIS) in case of a testis and gonadoblastoma (GB) in the DSD gonad. The actual risk for malignant transformation in individual DSD patients is hard to predict, because of confusing terminology referring to the different forms of DSD, and unclear criteria for identification of the presence of malignant germ cells, especially in young patients. This is specifically due to the phenomenon of delay of germ cell maturation, which might result in over diagnosis. This review will give novel insight into the pathogenesis of the type II GCTs through the study of patients with various forms of DSD for which the underlying molecular defect is known. To allow optimal understanding of the pathogenesis of this type of cancers, first normal gonadal development, especially regarding the germ cell lineage, will be discussed, after which type II GCTs will be introduced. Subsequently, the relationship between type II GCTs and DSD will be described, resulting in a number of new insights into the development of the precursor lesions of these tumors.
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http://dx.doi.org/10.1016/j.mce.2008.02.028DOI Listing
September 2008

Further characterization of the first seminoma cell line TCam-2.

Genes Chromosomes Cancer 2008 Mar;47(3):185-96

Department of Pathology, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands.

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.
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http://dx.doi.org/10.1002/gcc.20520DOI Listing
March 2008

Chromosomes and expression in human testicular germ-cell tumors: insight into their cell of origin and pathogenesis.

Ann N Y Acad Sci 2007 Dec 2;1120:187-214. Epub 2007 Oct 2.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC-University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

Human germ-cell tumors (GCTs) are a heterogeneous group of neoplasms. Based on epidemiology, anatomical site of presentation, histology, chromosomal constitution, and pattern of genomic imprinting, GCTs are classified into five entities. Within the testis, three types of GCTs can be diagnosed: type I (teratomas and yolk-sac tumors of neonates and infants); type II (seminomas and nonseminomas); type III (spermatocytic seminomas). Here the focus is on the type II GCTs, the most frequent type in the adult testis (so-called TGCTs). They can also be diagnosed in dysgenetic gonads (an incomplete or defective formation of the gonad, caused by a disturbed process of migration of the germ cells and/or their correct organization in their fetal gonadal ridge), the anterior mediastinum, and pineal/suprasellar region. In the testis, they originate from the malignant counterpart of primordial germ cells/gonocytes, referred to as carcinoma in situ (CIS)/intratubular germ-cell neoplasia unclassified (ITGCNU). CIS/ITGCNU and seminomatous cells are characterized by expression of OCT3/4 and NANOG, while in addition embryonal carcinoma expresses SOX2, all identified as transcription factors related to pluripotency in embryonic stem (ES) cells. With the exception of teratomas, most histological elements of TGCTs are sensitive for (cisplatin-based) chemotherapy; CIS/ITGCNU and seminoma cells are also sensitive to DNA damage induced by irradiation. Similar observations have been made for ES cells and their derivates. Moreover, the genetic constitution of TGCTs (low incidence of mutations and frequent uniparental disomies) can also be linked to characteristics of ES cells, likely related to their specific inability to repair DNA damage and their high sensitivity to apoptotic cell death. The unusual presence of wild-type P53 in TGCTs is explained by specific expression of a cluster of micro-RNAs (miRNAs), that is, hsa-miR 371-373, also expressed in ES cells, which prevents P53-driven cellular senescence upon oncogenic stress. Many characteristics of human TGCTs reflect the nonmalignant counterparts from which they originate. Demonstration of these characteristics, in combination with the knowledge of the abnormal niche of these cells, normally occupied by spermatogonia, allows an informative method for (early) diagnosis. The conclusion is that TGCTs are embryonic cancers found in adults.
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http://dx.doi.org/10.1196/annals.1411.000DOI Listing
December 2007

Tumor risk in disorders of sex development (DSD).

Best Pract Res Clin Endocrinol Metab 2007 Sep;21(3):480-95

Department of Pathology, Erasmus MC - University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Building Be, Room 430b, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.

Disorders of sex development (DSD), previously referred to as intersex disorders, comprise a variety of anomalies defined by congenital conditions in which chromosomal, gonadal, or anatomical sex is atypical. Besides issues such as gender assignment, clinical and diagnostic evaluation, surgical and psychosocial management, and sex steroid replacement, the significantly increased risk for developing specific types of malignancies is both clinically and biologically relevant. This relates to germ-cell tumors specifically in DSD patients with hypovirilization or gonadal dysgenesis. The presence of a well-defined part of the Y chromosome (known as the GBY region) is a prerequisite for malignant transformation, for which the testis-specific protein on the Y chromosome (TSPY) is a likely candidate gene. The precursor lesions of these cancers are carcinoma in situ (CIS)/intratubular germ-cell neoplasia unclassified (ITGCNU) in testicular tissue and gonadoblastoma in those without obvious testicular differentiation. Most recently, undifferentiated gonadal tissue (UGT) has been identified as the likely precursor for gonadoblastoma. The availability of markers for the different developmental stages of germ cells allows detailed investigation of the characteristics of normal and (pre)malignant germ cells. Although informative in a diagnostic setting for adult male patients, these markers - such as OCT3/4 - cannot easily distinguish (pre)malignant germ cells from germ cells showing delayed maturation. This latter phenomenon is frequently found in gonads of DSD patients, and may be related to the risk of malignant transformation. Thus, the mere application of these markers might result in over-diagnosis and unnecessary gonadectomy. It is proposed that morphological and histological evaluation of gonadal tissue, in combination with OCT3/4 and TSPY double immunohistochemistry and clinical parameters, is most informative in estimating the risk for germ-cell tumor development in the individual patient, and might in future be used to develop a decision tree for optimal management of patients with DSD.
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http://dx.doi.org/10.1016/j.beem.2007.05.001DOI Listing
September 2007

Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines.

Genes Chromosomes Cancer 2007 Oct;46(10):895-908

Department of Pathology, Erasmus Medical Center, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Rotterdam, The Netherlands.

Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the U133Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity.
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http://dx.doi.org/10.1002/gcc.20475DOI Listing
October 2007

Genomic and expression profiling of human spermatocytic seminomas: pathogenetic implications.

Int J Androl 2007 Aug 15;30(4):328-35; discussion 335-6. Epub 2007 Jun 15.

Department of Pathology, Erasmus MC - University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Rotterdam, The Netherlands.

Traditionally, germ cell tumours (GCTs) have been classified based on their histological appearance, i.e. the differentiation of lineages. Various subtypes of seminomas have been identified in a number of classification systems, including anaplastic and spermatocytic seminoma. Here we present conclusive evidence that spermatocytic seminomas are GCTs with a separate pathogenesis from seminoma, and should therefore not be considered as a variant of seminoma. Moreover, the different putative animal models, either spontaneous or induced, are discussed in the context of their value as model for human GCTs. The canine seminomas are currently considered as the most informative model for human spermatocytic seminomas.
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http://dx.doi.org/10.1111/j.1365-2605.2007.00779.xDOI Listing
August 2007

Novel FXXFF and FXXMF motifs in androgen receptor cofactors mediate high affinity and specific interactions with the ligand-binding domain.

J Biol Chem 2006 Jul 11;281(28):19407-16. Epub 2006 May 11.

Department of Urology, Josephine Nefkens Institute, Erasmus MC, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands.

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for alpha-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.
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http://dx.doi.org/10.1074/jbc.M602567200DOI Listing
July 2006

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping.

Mol Endocrinol 2006 Aug 20;20(8):1742-55. Epub 2006 Apr 20.

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.
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http://dx.doi.org/10.1210/me.2005-0348DOI Listing
August 2006

Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene.

Cancer Res 2006 Jan;66(1):290-302

Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center/University Medical Center, Rotterdam, The Netherlands.

Spermatocytic seminomas are solid tumors found solely in the testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and numerical chromosomal changes through conventional karyotyping, spectral karyotyping, and array comparative genomic hybridization using a 32 K genomic tiling-path resolution BAC platform (confirmed by in situ hybridization). Our panel of five spermatocytic seminomas showed a specific pattern of chromosomal imbalances, mainly numerical in nature (range, 3-24 per tumor). Gain of chromosome 9 was the only consistent anomaly, which in one case also involved amplification of the 9p21.3-pter region. Parallel chromosome level expression profiling as well as microarray expression analyses (Affymetrix U133 plus 2.0) was also done. Unsupervised cluster analysis showed that a profile containing transcriptional data on 373 genes (difference of > or = 3.0-fold) is suitable for distinguishing these tumors from seminomas/dysgerminomas. The diagnostic markers SSX2-4 and POU5F1 (OCT3/OCT4), previously identified by us, were among the top discriminatory genes, thereby validating the experimental set-up. In addition, novel discriminatory markers suitable for diagnostic purposes were identified, including Deleted in Azospermia (DAZ). Although the seminomas/dysgerminomas were characterized by expression of stem cell-specific genes (e.g., POU5F1, PROM1/CD133, and ZFP42), spermatocytic seminomas expressed multiple cancer testis antigens, including TSP50 and CTCFL (BORIS), as well as genes known to be expressed specifically during prophase meiosis I (TCFL5, CLGN, and LDHc). This is consistent with different cells of origin, the primordial germ cell and primary spermatocyte, respectively. Based on the region of amplification defined on 9p and the associated expression plus confirmatory immunohistochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for involvement in the development of spermatocytic seminomas.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-2936DOI Listing
January 2006
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