Variations of sex development: The first German interdisciplinary consensus paper.

INTRODUCTION: The term variations of sex development subsumes a large number of congenital conditions including chromosomal mosaics and variations of chromosomal, gonadal, and phenotypic sex. A situation of this nature may cause severe distress to both, parents and affected persons. One of the reasons for this is the binary form of gender classification in the society. In the past, because of a fear of possible stigmatization and an inability to cope with complex situations, it has been medical policy and practice for newborns to undergo early, mostly 'feminizing' elective surgery with the aim of achieving an outer genital appearance that is unambiguously male or female. Protests by advocacy groups for the most part as well as the results of outcome studies have shown that the development of affected persons may be very different to what has been expected and often does not result in the intended clear female or male gender identity as had been intended. It, therefore, seemed a matter of urgency to implement this new awareness as well as the ethical and personal human rights perspectives in the recommendations for the medical and psychosocial management of diverse sex development (DSD) in the future. STUDY DESIGN: In 2012, an interdisciplinary group of German academics engaged in the field of DSD decided to work on a consensus paper for this topic. It involved the participation of all faculties and non-scientific groups dealing with DSD, in particular advocacy and service-user groups. In a structured consensus, process recommendations were developed based on scientific literature as well as personal experiences of clinicians and affected individuals. RESULTS: Finally, 37 recommendations were agreed on. The strength of consensus is reflected in the degree of agreement as expressed in percentages. CONCLUSION: The introduction of the consensus paper reflects on the emerging paradigm shift and the necessity for a more open view of gender within society. The paper is intended to aid the performance of appropriate diagnostics in DSD-affected newborns and especially to help parents and affected persons cope with the biological and social consequences of DSD. With regard to medical or surgical therapy, it gives information about the most recent treatment trends.

Array-comparative genomic hybridization analysis in patients with Müllerian fusion anomalies.

Fusion anomalies of the Müllerian ducts are associated with an increased risk for miscarriage and premature labor. In most cases polygenic-multifactorial inheritance can be assumed but autosomal-dominant inheritance with reduced penetrance and variable manifestation should be considered. We performed array-comparative genomic hybridization (CGH) analysis in a cohort of 103 patients with Müllerian fusion anomalies. In 8 patients we detected microdeletions and microduplications in chromosomal regions 17q12, 22q11.21, 9q33.1, 3q26.11 and 7q31.1. The rearrangement in 17q12 including LHX1 and HNF1ß as well as in 22q11.21 have already been observed in MRKHS (Mayer-Rokitansky-Küster-Hauser syndrome). In summary, we (1) detected causative micro-rearrangements in patients with Müllerian fusion anomalies, (2) show that Müllerian fusion anomalies and MRKHS may have a common etiology, and (3) identified new candidate genes for Müllerian fusion anomalies.

Mutations in WNT9B are associated with Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a well-known malformation pattern of the Müllerian ducts (MDs) characterized by congenital absence of the uterus and vagina. To date, most cases remain unexplained at molecular level. As female Wnt9b-/- mice show a MRKHS-like phenotype, WNT9B has emerged as a promising candidate gene for this disease. We performed retrospective sequence analyses of WNT9B in 226 female patients with disorders of the MDs, including 109 patients with MRKHS, as well as in 135 controls. One nonsense mutation and five likely pathogenic missense mutations were detected in WNT9B. Five of these mutations were found in cases with MRKHS accounting for 4.6% of the patients with this phenotype. No pathogenic mutations were detected in the control group (p = 0.017). Interestingly, all of the MRKHS patients with a WNT9B mutation were classified as MRKHS type 1, representing 8.5% of the cases from this subgroup. In previous studies, two of the patients with a WNT9B mutation were found to carry either an additional deletion of LHX1 or a missense mutation in TBX6. We conclude that mutations in WNT9B were frequently associated with MRKHS in our cohort and some cases may be explained by a digenic disease model.

Experts' Opinion on the Prenatal Therapy of Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency - Guideline of DGKED in cooperation with DGGG (S1-Level, AWMF Registry No. 174/013, July 2015).

Purpose: This guideline of the German Society of Pediatric Endocrinology and Diabetology (DGKED) is designed to be experts' opinion on the current concept of prenatal therapy for congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH). Several scientific medical societies have also participated in the guideline. It aims to offer guidance to physicians when they counsel affected families about prenatal therapy. Methods: The experts commissioned by the medical societies developed a consensus in an informal process. The consensus was subsequently confirmed by the steering committees of the respective medical societies. Recommendations: Prenatal CAH therapy is an experimental therapy. We recommend designing and using standardized protocols for the prenatal diagnosis, therapy and long-term follow-up of women and children treated prenatally with dexamethasone. If long-term follow-up is not possible, then prenatal therapy should not be performed.

[Genetic aspects of fertility disorders]. / Genetische Aspekte bei Störungen der Fertilität.

Genetic disorders of fertility can occur at the level of gonadal differentiation or function, germ cell production or function, and the genital ducts. In gonadal dysgenesis, the differentiation of testes or ovaries is impaired. Gonadal dysgenesis can be caused by chromosome aberrations or monogenetic defects in XY or XX gonadal dysgenesis. For the biosynthesis of sexual hormones, a normal development of the gonads and an intact hypothalamic-pituitary-gonadal axis is necessary. Disorders of steroid hormone synthesis are associated with an increased or diminished production of sexual hormones. Clinical and genetic aspects of adrenogenital syndrome are discussed here. Mutations of the androgen receptor cause a spectrum of androgen insensitivity ranging from women with female external genitalia through patients with genital ambiguity to men with infertility. Disturbed spermatogenesis is heterogeneous and can be the result of chromosome aberrations such as Klinefelter syndrome or structural aberrations as translocations and microdeletions of the Y chromosome. Premature ovarian failure is characterized by amenorrhea and hypergonadotropic hypogonadism before 40 years of age. Beside nongenetic factors, premature ovarian failure can be caused by chromosome aberrations or monogenetic defects. Disorders of the genital ducts such as anomalies of the müllerian ducts in females and of the wolffian ducts in males can be associated with sterility or infertility.

CD34(+) lineage specific donor cell chimerism for the diagnosis and treatment of impending relapse of AML or myelodysplastic syndrome after allo-SCT.

After allo-SCT, analysis of CD34(+) lineage-specific donor cell chimerism (DCC) is a sensitive method for monitoring minimal residual disease in patients with AML or myelodysplastic syndrome (MDS) with CD34 expression. To substantiate evidence of whether immune interventions in patients with impending relapse, defined by incomplete lineage-specific DCC, may prevent hematological relapse, we performed a retrospective nested case control study. Unsorted and lineage-specific DCC were measured in 134 patients. Forty-three patients had an incomplete CD34(+)-DCC with no other evidence of relapse. After immediate tapering of immunosuppressive treatment (30 patients) and/or infusion of donor lymphocytes (10 patients), 21 patients remained in remission (conversion to complete lineage-specific DCC) and 22 relapsed. Relapse-free survival at 3 years of the 91 patients with stable DCC and of the 43 patients with incomplete DCC was 74% (95% confidence interval (CI), 64-83%) and 40% (95% CI, 24-58%), respectively. OS rates were 79% (95% CI, 70-88%) and 52% (95% CI, 35-69%), respectively. These results, with 49% of patients with impending relapse successfully treated with immune intervention, highly suggest that analysis of CD34(+)-DCC is an important tool for monitoring and the management of AML and MDS patients after allo-SCT.

Frame shift mutation of LHX1 is associated with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome.

BACKGROUND: The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the uterus and the upper part of the vagina in women who usually have normal ovaries and a 46, XX karyotype. MRKH can occur as an isolated form (type I) or in combination with various malformations as a syndromic or a type II MRKH. To date, in most of the cases the underlying etiology remains unclear. Recently, in approximately 6% of MRKH patients, deletions of chromosomal region 17q12 have been identified. The LHX1 gene, which is located in the deletion interval, has been suggested to be a strong candidate, because targeting inactivation of Lhx1 causes a complex phenotype including aplasia of the Müllerian ducts. METHODS AND RESULTS: By sequence analysis of LHX1 in a large cohort of MRKH patients, we detected a heterozygous frame shift mutation resulting in a premature stop codon. Previously, we have reported a heterozygous missense mutation of LHX1 in another MRKH patient. CONCLUSIONS: We conclude that heterozygous mutations of LHX1 might be one cause of the MRKH syndrome in a subgroup of patients.

BRCA1/2 testing: uptake, phenocopies, and strategies to improve detection rates in initially negative families.

In families with clustering of breast and ovarian cancer, molecular testing of the major susceptibility genes BRCA1/2 helps to identify patients with disease mutations and healthy persons at high risk who can participate in targeted intervention programs. We investigated 5559 families from the German Consortium for Hereditary Breast and Ovarian Cancer included between 1997 and 2008 and treated under clinical routine conditions. In each family an index patient/person had been screened for deleterious mutations in BRCA1/2. Healthy relatives agreed to predictive testing in 888 of 1520 BRCA1/2 mutation-positive families (58%). Of 2646 eligible unaffected first-degree relatives 1143 decided to be tested (43%). In 325 families with BRCA1/2-positive index patients one related BC/OC patient was tested and 39 (12.0%; 95% confidence interval: 8.7-16.0%) discrepant cases found. A second related individual was screened in 163 of 3388 (4.9%) families with BRCA1/2-negative index patient and in eight families a BRCA1/2 mutation was found. In BRCA1/2 mutation-positive families, BC/OC patients lacking the familial mutation have to be expected at a rather high rate. In families with BRCA1/2-negative index patient we recommend a second screening if another patient with a high probability of carrying a BRCA1/2 mutation is available.

Bcar3 is expressed in sertoli cells and germ cells of the developing testis in mice.

We identified Bcar3 in the course of a screen for developmentally regulated genes at early developmental stages in mouse embryos. In this study, we explored the spatio-temporal expression pattern of Bcar3 during the critical time period of sex determination using in situ hybridization, real-time RT-PCR, and immunohistochemistry. We found that Bcar3 is expressed in XY gonads during early stages of gonad development and that BCAR3 localizes to Sertoli cells and germs cells. In addition, we identified a new alternative Bcar3 transcript in which exons 4-7 are deleted. This deletion could result in the generation of a truncated BCAR3 protein lacking functional domains including the SH2 domain. The data presented here suggest that Bcar3 could play a role in gonad development.

Analysis of the Wilms' tumor suppressor gene (WT1) in patients 46,XY disorders of sex development.

CONTEXT: The Wilms' tumor suppressor gene (WT1) is one of the major regulators of early gonadal and kidney development. WT1 mutations have been identified in 46,XY disorders of sex development (DSD) with associated kidney disease and in few isolated forms of 46,XY DSD. OBJECTIVE: The objective of the study was the evaluation of WT1 mutations in different phenotypes of isolated 46,XY DSD and clinical consequences. DESIGN: The design of the study was: 1) sequencing of the WT1 gene in 210 patients with 46,XY DSD from the German DSD network, consisting of 150 males with severe hypospadias (70 without cryptorchidism, 80 with at least one cryptorchid testis), 10 males with vanishing testes syndrome, and 50 raised females with partial to complete 46,XY gonadal dysgenesis; and 2) genotype-phenotype correlation of our and all published patients with 46,XY DSD and WT1 mutations. RESULTS: We have detected WT1 mutations in six of 80 patients with severe hypospadias (7.5%) and at least one cryptorchid testis and in one of 10 patients with vanishing testes syndrome (10%). All patients except one developed Wilms' tumor and/or nephropathy in childhood or adolescence. CONCLUSION: WT1 analysis should be performed in newborns with complex hypospadias with at least one cryptorchid testis and in isolated 46,XY partial to complete gonadal dysgenesis. Kidney disease might not develop until later life in these cases. WT1 analysis is mandatory in all 46,XY DSD with associated kidney disease. WT1 analysis is not indicated in newborns with isolated hypospadias without cryptorchidism. Patients with WT1 mutations should be followed up closely because the risk of developing a Wilms' tumor, nephropathy, and/or gonadal tumor is very high.

Mutations of the SRY-responsive enhancer of SOX9 are uncommon in XY gonadal dysgenesis.

During mouse sex determination, SRY upregulates the core testis-specific enhancer of Sox9, TESCO. Mutations in human SRY are found in one third of cases with XY pure gonadal dysgenesis (XY GD; Swyer syndrome), while two thirds remain unexplained. Heterozygous SOX9 mutations can cause XY GD in association with the skeletal malformation syndrome campomelic dysplasia. We hypothesized that human TESCO mutations could cause isolated XY GD. Sixty-six XY GD cases with an intact SRY were analyzed for TESCO point mutations or deletions. No mutations were identified. We conclude that TESCO mutations are not a common cause of XY GD.

New technologies for the identification of novel genetic markers of disorders of sex development (DSD).

Although the genetic basis of human sexual determination and differentiation has advanced considerably in recent years, the fact remains that in most subjects with disorders of sex development (DSD) the underlying genetic cause is unknown. Where pathogenic mutations have been identified, the phenotype can be highly variable, even within families, suggesting that other genetic variants are influencing the expression of the phenotype. This situation is likely to change, as more powerful and affordable tools become widely available for detailed genetic analyses. Here, we describe recent advances in comparative genomic hybridisation, sequencing by hybridisation and next generation sequencing, and we describe how these technologies will have an impact on our understanding of the genetic causes of DSD.

Array-CGH analysis in patients with syndromic and non-syndromic XY gonadal dysgenesis: evaluation of array CGH as diagnostic tool and search for new candidate loci.

BACKGROUND: XY gonadal dysgenesis (XY-GD) is a heterogeneous disorder characterized by failure of testicular development despite a normal male karyotype. Non-syndromic and syndromic forms can be delineated. Currently, only a minority of cases can be explained by gene mutations. METHODS: The aim of this study was to detect microdeletions and duplications by using high-resolution Agilent oligonucleotide arrays in a cohort of 87 patients with syndromic or non-syndromic 46,XY-GD. RESULTS: In 26 patients, we identified gains or losses in regions including genes involved in XY-GD (DMRT1, SOX9, DAX1) or in regions, which have not been described as polymorphic copy number variants (CNVs). CONCLUSIONS: This study shows that array comparative genomic hybridization (CGH) analysis is a useful tool for the molecular diagnosis of XY-GD as well as for the identification of potential candidate genes involved in male sexual development.

Copy number variants in premature ovarian failure and ovarian dysgenesis.

Premature ovarian failure (POF) is a heterogeneous group of disorders with amenorrhea and high serum gonadotropins in women of less than 40 years. Ovarian dysgenesis (OD) which is characterised by the loss of follicles before puberty describes the most severe POF outcome. Although a multitude of different factors including non-genetic as well as genetic causes are known to play a role in the development of POF and OD, the underlying etiology remains unsolved in the majority of cases. In the last years, array-CGH was found to be a very useful tool in the identification of candidate genes in different conditions. Therefore, we performed array-CGH analysis by using high-resolution Agilent oligonucleotide arrays in a total of 74 POF and OD patients and identified 44 private losses and gains potentially causative for POF. It is striking to note that a lot of the genes involved in these rearrangements can be classified in (i) genes involved in meiosis (e.g. PLCB1, RB1CC1, MAP4K4), (ii) genes involved in DNA repair (e.g. RBBP8) and (iii) genes involved in folliculogenesis or male fertility in homologs of model organisms (e.g. IMMP2L, FER1L6, MEIG1).

Blood chimerism in a girl with Down syndrome and possible freemartin effect leading to aplasia of the Müllerian derivatives.

Cytogenetic and molecular genetic analysis in a case of sex-discordant dizygotic twins revealed blood chimerism in the girl (46,XY in blood and 47,XX, + 21 in fibroblasts) caused by feto-fetal transfusion from her healthy brother. The girl presented with Down syndrome, aplasia of the uterus and the Fallopian tubes and normal female external genitalia. We propose that the lack of Müllerian structures is caused by the effect of the Müllerian inhibiting substance transferred from the male to the female twin in early pregnancy. This disorder of sex development is known as freemartin phenomenon in female cattle from sex-discordant twin pairs.

Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function.

BACKGROUND: Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. OBJECTIVE: To characterise genetic and clinical findings in patients with SIX3 mutations. METHODS: Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. RESULTS: In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. CONCLUSIONS: Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype-phenotype correlation, as shown by functional studies using animal models.

PLAG1 activation in lipoblastoma coinciding with low-level amplification of a derivative chromosome 8 with a deletion del(8)(q13q21.2).

Lipoblastoma is a benign uncommon soft-tissue-tumor resembling fetal adipose tissue affecting mainly children under three years of age. In lipoblastoma, the typical cytogenetic changes are clonal rearrangements involving chromosomal region 8q11-->q13. The oncogene PLAG1 (pleomorphic adenoma gene 1) is located within this chromosomal region on band 8q12. Recent reports have demonstrated that in lipoblastoma, the PLAG1 gene is activated by 'promoter-swapping'. Herein, we demonstrate that in lipoblastoma, the PLAG1 gene may also be activated by low-level amplification. We report on a lipoblastoma with the karyotype 48 approximately 50,XX,del(8)(q13q21.2),+del(8)(q13q21.2)x4[cp12]. Subsequent FISH analysis on uncultured tumor cells confirmed this result and demonstrated a low-level amplification of the chromosomal region 8pter-->8q13 and 8q21.2-->8qter. A partial monosomy was seen for the chromosomal region 8q13-->8q21.2. No other gains or losses were observed by CGH analysis. RT-PCR analysis showed that the PLAG1 gene is activated in the tumor sample of the lipoblastoma analyzed, in contrast to normal fatty tissue without PLAG1 expression. In conclusion, our results demonstrate that low-level amplification is a further mechanism of PLAG1 activation in lipoblastomas.