New genomic technologies: an aid for diagnosis of disorders of sex development.

The Chicago Consensus Conference of 2005 defined Disorders of Sex Development (DSD) as "congenital conditions in which the development of chromosomal, gonadal or anatomic sex is atypical." DSD diagnoses are difficult to establish. A lack of standardization of anatomical and endocrine phenotyping and the limited number of known DSD genes and genotype/correlation has long hampered the field, leaving many patients without a definitive diagnosis. The resulting uncertainty may intrinsically pose a great amount of discomfort to affected individuals and their families. DSD-causative genes have historically been identified thanks to positional cloning of disease-associated variants segregating in families or chromosomal rearrangements. Recent advances of chromosomal microarray and exome sequencing technologies are allowing for higher rates of diagnostic success for DSD patients and are changing clinical practice. In this review, we discuss the application of these technologies and their findings as an upcoming model for clinical diagnosis of DSD. We show that exome sequencing is a valuable tool and we propose that it should be used as a first-stage diagnostic technique because it allows for early identification of a genetic cause that may be critical for patient management.

Oxidative stress-mediated iNKT-cell activation is involved in COPD pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a major clinical challenge mostly due to cigarette smoke (CS) exposure. Invariant natural killer T (iNKT) cells are potent immunoregulatory cells that have a crucial role in inflammation. In the current study, we investigate the role of iNKT cells in COPD pathogenesis. The frequency of activated NKT cells was found to be increased in peripheral blood of COPD patients relative to controls. In mice chronically exposed to CS, activated iNKT cells accumulated in the lungs and strongly contributed to the pathogenesis. The detrimental role of iNKT cells was confirmed in an acute model of oxidative stress, an effect that depended on interleukin (IL)-17. CS extracts directly activated mouse and human dendritic cells (DC) and airway epithelial cells (AECs) to trigger interferonγ and/or IL-17 production by iNKT cells, an effect ablated by the anti-oxidant N-acetylcystein. In mice, this treatment abrogates iNKT-cell accumulation in the lung and abolished the development of COPD. Together, activation of iNKT cells by oxidative stress in DC and AECs participates in the development of experimental COPD, a finding that might be exploited at a therapeutic level.

Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development.

Disorders of sex development (DSD) are rare disorders in which there is discordance between chromosomal, gonadal, and phenotypic sex. Only a minority of patients clinically diagnosed with DSD obtains a molecular diagnosis, leaving a large gap in our understanding of the prevalence, management, and outcomes in affected patients. We created a novel DSD-genetic diagnostic tool, in which sex development genes are captured using RNA probes and undergo massively parallel sequencing. In the pilot group of 14 patients, we determined sex chromosome dosage, copy number variation, and gene mutations. In the patients with a known genetic diagnosis (obtained either on a clinical or research basis), this test identified the molecular cause in 100% (7/7) of patients. In patients in whom no molecular diagnosis had been made, this tool identified a genetic diagnosis in two of seven patients. Targeted sequencing of genes representing a specific spectrum of disorders can result in a higher rate of genetic diagnoses than current diagnostic approaches. Our DSD diagnostic tool provides for first time, in a single blood test, a comprehensive genetic diagnosis in patients presenting with a wide range of urogenital anomalies.

The endless quest for sex determination genes.

Disorders in human sex determination cause defects in gonadal function and can result in a spectrum of abnormalities in the internal and external genitalia, ranging from relatively mild sexual ambiguities to complete sex reversal. Several genes involved in sex determination have been validated in humans, and activities of their gene products are being elucidated, particularly in mouse models. However, how these genes interact in an overall process remains far from clear, and it is probable that many additional genes are involved. Management of patients with pathologies in sex determination and subsequent differentiation is currently under debate, but will require not only an understanding of the multiple definitions of an individual's sex but also an increased knowledge of the molecular mechanisms involved in sex determination.

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients.

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.

Adrenal hypoplasia congenita with multiple pituitary hormone deficiency without documented mutation in DAX1 or SF1 gene.

A boy with adrenal hypoplasia congenita (AHC) and multiple pituitary hormone deficiency (MPHD), without mutations in the DAX1 or SF1 genes, is described. The association of AHC and MPHD has not been previously reported.

Up-regulation of WNT-4 signaling and dosage-sensitive sex reversal in humans.

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

Primate DAX1, SRY, and SOX9: evolutionary stratification of sex-determination pathway.

The molecular evolution of DAX1, SRY, and SOX9, genes involved in mammalian sex determination, was examined in six primate species. DAX1 and SRY have been added to the X and Y chromosomes, respectively, during mammalian evolution, whereas SOX9 remains autosomal. We determined the genomic sequences of DAX1, SRY, and SOX9 in all six species, and calculated K(a), the number of nonsynonymous substitutions per nonsynonymous site, and compared this with the K(s), the number of synonymous substitutions per synonymous site. Phylogenetic trees were constructed by means of the DAX1, SRY, and SOX9 coding sequences, and phylogenetic analysis was performed using maximum likelihood. Overall measures of gene and protein similarity were closer for DAX1 and SOX9, but DAX1 exhibited nonsynonymous amino acid substitutions at an accelerated frequency relative to synonymous changes, similar to SRY and significantly higher than SOX9. We conclude that, at the protein level, DAX1 and SRY are under less selective pressure to remain conserved than SOX9, and, therefore, diverge more across species than does SOX9. These results are consistent with evolutionary stratification of the mammalian sex determination pathway, analogous to that for sex chromosomes.

AluY insertion (IVS4-52ins316alu) in the glycerol kinase gene from an individual with benign glycerol kinase deficiency.

Glycerol kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated glycerol kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subject's glycerol kinase gene showed the insertion of an AluY sequence in intron 4 of the glycerol kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of glycerol kinase deficiency caused by an Alu insertion.

Midkine is expressed early in rat fetal adrenal development.

Adrenal gland development is complex and poorly understood at the molecular level. Only a subset of patients with adrenal hypoplasia congenita (AHC) carry mutations in DAX1, a member of the nuclear hormone receptor superfamily. Therefore we set out to identify other candidate genes responsible for AHC by characterizing genes involved in fetal adrenal development. To identify these genes, we studied the differential expression of genes in fetal rat adrenals comparing tissues at 14 and 15 days postcoitum (dpc) since this period encompasses major morphological change in rat adrenal development. Fetal rat adrenals were dissected, cDNAs were prepared, and suppressive subtractive hybridization was performed. We isolated 126 clones of putatively differentially expressed clones and approximately 250 bp of each of the clones was sequenced. The most interesting putative developmental genes were examined. One member of the extracellular PTN/MDK (pleiotrophin/midkine) heparin-binding protein family involved in regulation of growth and differentiation was selected for initial study. We obtained full-length transcript by 3' rapid amplification of cDNA ends and performed Northern analysis on rat adrenal RNA from fetuses at 13, 14, 15, 17, and 19 dpc and newborns. Results from those analyses demonstrated the highest Mdk expression at days 13 and 14 followed by a moderate decrease of expression during the fetal stages thereafter. In the newborn, Mdk expression is nearly undetectable. Our results indicate that Mdk has a very specific pattern of fetal expression in the adrenals. We conclude that Mdk is involved early in fetal development of the rat adrenal. Therefore, MDK is a candidate gene for AHC not due to DAX1 mutations.

Genetics of sexual development.

In humans, the choice between male or female development is genetically determined. Sex determination occurs as the undifferentiated and bipotential embryonic gonad becomes either testes or ovary. This process is influenced by the action of genes that have been discovered by genetics studies of sex-reversed patients, XY females, XX males, and XX true hermaphrodites. The development of the gonad is better known in males than in females. SRY, a gene located on the Y chromosome, triggers a complex genetic cascade leading to testicular development. Other genes such as DAX1, located on the X chromosome, antagonize the action of SRY and may influence ovarian development. Only a minority of sex-reversed patients can be explained genetically, suggesting that many genes influencing sex determination are yet to be discovered.

IMAGe, a new clinical association of intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies.

We report three boys with adrenal hypoplasia congenita (AHC) and additional findings that represent a new syndrome, IMAGe: Intrauterine growth retardation, Metaphyseal dysplasia, AHC, and Genital anomalies. Each presented shortly after birth with growth retardation and severe adrenal insufficiency. Each of the three patients had mild dysmorphic features, bilateral cryptorchidism, a small penis, and hypogonadotropic hypogonadism. Skeletal surveys revealed metaphyseal dysplasia in all three and epiphyseal dysplasia in two. The patients had documented or suspected hypercalciuria and/or hypercalcemia, resulting in nephrocalcinosis in one and in prenatal liver and spleen calcifications in another. AHC presents most often either as an isolated abnormality, caused by mutations in the DAX1 gene, or as part of an Xp21 contiguous gene syndrome, caused by a deletion of the Duchenne muscular dystrophy, glycerol kinase, and DAX1 genes. All three patients with the IMAGe association had normal creatine kinase levels and no evidence of glycerol kinase deficiency. Sequence analysis of DNA from these patients revealed no mutation in the DAX1- or steroidogenic factor-1-coding sequences, nor was a deletion of DAX1 detected. Identification of the molecular basis of the IMAGe association will give new insight into the pathogenesis of this syndromic relationship involving bone, adrenal cortical, and pituitary development.

Mammalian sex determination: from gonads to brain.

In mammals, sex is determined by the Y chromosome, which encodes a testis-determining factor (TDF). This factor causes the undifferentiated embryonic gonads to develop as testes rather than ovaries. The testes subsequently produce the male sex hormones that are responsible for all male sexual characteristics. In 1990, the sex-determining gene, TDF, was identified and termed SRY in humans (Sry in mice). It encodes a protein containing a high mobility group (HMG) motif, which confers the ability to bind and to bend DNA. Genetic evidence supporting SRY as TDF came from the observation of a male phenotype in XX mice transgenic for a small genomic fragment containing Sry, and from the study of XY sex-reversed individuals who harbor de novo mutations in the SRY coding sequence. Other non-Y-linked genes involved in sex determination were subsequently found by genetic analysis of XY sex-reversed patients not explained by mutations in SRY. These genes are WT1, SF1, DAX1, and SOX9. A regulatory cascade hypothesis for mammalian sex determination, proposing that SRY represses a negative regulator of male development, was recently supported by observation of mice that expressed a DAX1 transgene and developed as XY sex-reversed females. The role of some sex-determining genes, such as DAX1 and SF1, in the development of the entire reproductive axis, a functionally integrated endocrine axis, leads to a new concept. Normal sexual development may result from the functional and developmental integration of a number of different genes that play roles in sex determination, sexual differentiation, and sexual behavior.

DAX1 mutations map to putative structural domains in a deduced three-dimensional model.

The DAX1 protein is an orphan nuclear hormone receptor based on sequence similarity in the putative ligand-binding domain (LBD). DAX1 mutations result in X-linked adrenal hypoplasia congenita (AHC). Our objective was to identify DAX1 mutations in a series of families, to determine the types of mutations resulting in AHC and to locate single-amino-acid changes in a DAX1 structural model. The 14 new mutations identified among our 17 families with AHC brought the total number of families with AHC to 48 and the number of reported mutations to 42; 1 family showed gonadal mosaicism. These mutations included 23 frameshift, 12 nonsense, and six missense mutations and one single-codon deletion. We mapped the seven single-amino-acid changes to a homology model constructed by use of the three-dimensional crystal structures of the thyroid-hormone receptor and retinoid X receptor alpha. All single-amino-acid changes mapped to the C-terminal half of the DAX1 protein, in the conserved hydrophobic core of the putative LBD, and none affected residues expected to interact directly with a ligand. We conclude that most genetic alterations in DAX1 are frameshift or nonsense mutations and speculate that the codon deletion and missense mutations give insight into the structure and function of DAX1.

DAX1 gene expression upregulated by steroidogenic factor 1 in an adrenocortical carcinoma cell line.

Two nuclear hormone receptor superfamily members, DAX1 and SF1, are required for normal adrenal cortical development. Mutations in DAX1 are responsible for X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism. Steroidogenic Factor 1 (SF1) regulates the expression of a number of steroidogenic genes and a putative SF1 response element (SF1-RE) in the DAX1 promoter which binds SF1 specifically. Therefore, we examined deletions in the DAX1 promoter driving expression of beta-galactosidase, with and without coexpression of SF1, in the human adrenocortical carcinoma cell line NCI-H295. We defined the DAX initiation start site and localized the putative SF1-RE at -135 to -143 bp. Loss of the putative SF1-RE region or specific removal of the 9-bp SF1 site resulted in decreased transcriptional activity by 2.3-to 2.5-fold. When cotransfected with 1550 bp of the DAX1 promoter, an SF1-containing expression vector increased the transcriptional activity of the DAX1 promoter by 4-fold. No significant change above baseline occurred when the cells were cotransfected with the 1541-bp fragment containing the entire 1550-bp promoter region minus the 9-bp SF1-RE. We conclude that the SF1-RE is an enhancer element within the DAX1 promoter and speculate that SF1 may be a transcription factor that acts, at least in part, through DAX1 for normal adrenal cortical development.

Loss of sequences 3' to the testis-determining gene, SRY, including the Y pseudoautosomal boundary associated with partial testicular determination.

The condition termed 46,XY complete gonadal dysgenesis is characterized by a completely female phenotype and streak gonads. In contrast, subjects with 46,XY partial gonadal dysgenesis and those with embryonic testicular regression sequence usually present ambiguous genitalia and a mix of Müllerian and Wolffian structures. In 46,XY partial gonadal dysgenesis gonadal histology shows evidence of incomplete testis determination. In 46,XY embryonic testicular regression sequence there is lack of gonadal tissue on both sides. Various lines of evidence suggest that embryonic testicular regression sequence is a variant form of 46,XY gonadal dysgenesis. The sex-determining region Y chromosome gene (SRY) encodes sequences for the testis-determining factor. To date germ-line mutations in SRY have been reported in approximately 20% of subjects with 46,XY complete gonadal dysgenesis. However, no germ-line mutations of SRY have been reported in subjects with the partial forms. We studied 20 subjects who presented either 46,XY partial gonadal dysgenesis or 46,XY embryonic testicular regression sequence. We examined the SRY gene and the minimum region of Y-specific DNA known to confer a male phenotype. The SRY-open reading frame (ORF) was normal in all subjects. However a de novo interstitial deletion 3' to the SRY-ORF was found in one subject. Although it is possible that the deletion was unrelated to the subject's phenotype, we propose that the deletion was responsible for the abnormal gonadal development by diminishing expression of SRY. We suggest that the deletion resulted either in the loss of sequences necessary for normal SRY expression or in a position effect that altered SRY expression. This case provides further evidence that deletions of the Y chromosome outside the SRY-ORF can result in either complete or incomplete sex reversal.