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Study Question: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility?Summary Answer: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking.What Is Known Already: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure.Study Design, Size, Duration: A WES study was followed by flow cytometry studies of mutant protein function.Participants/materials, Setting, Methods: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry.Main Results and the Role Of Chance: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01).Limitations, Reasons For Caution: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant.Wider Implications Of the Findings: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine.Study Funding/competing Interests: E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose. [ABSTRACT FROM AUTHOR]

FOXL2 is a gene encoding a forkhead transcription factor, whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome that often involves premature ovarian failure. FOXL2 is one of the earliest ovarian markers and it offers, along with its targets, an excellent model to study ovarian development and function in normal and pathological conditions. We have recently shown that the aromatase gene is a target of FOXL2, and only three other targets have been reported so far. To detect potential transcriptional targets of FOXL2, we used DNA chips and quantitative PCR to compare the transcriptomes of granulosa-like cells overexpressing, or not, FOXL2. This analysis showed that mediators of inflammation, apoptotic and transcriptional regulators, genes involved in cholesterol metabolism, and genes encoding enzymes and transcription factors involved in reactive oxygen species detoxification were up-regulated. On the other hand, FOXL2 down-regulated the transcription of several genes involved in proteolysis and signal transduction and in transcription regulation. A bioinformatic analysis was conducted to discriminate between potential target promoters activated and repressed by FOXL2. In addition, the promoters of strongly activated genes were enriched in forkhead recognition sites, suggesting that these genes might be direct FOXL2 targets. Altogether, these results provide insight into the activity of FOXL2 and may help in understanding the mechanisms of pathogenesis of FOXL2 mutations if the targets prove to be the same in the ovary. [ABSTRACT FROM AUTHOR]

Objective Mutations in DAX-1, an X-linked gene encoding an orphan nuclear receptor, have been associated with adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Here we describe two novel DAX-1 mutations, Y214X and I361T, associated with childhood-onset primary adrenal failure. We aimed at analysing their effects on protein localization, transcriptional activity and propose a structural-function relationship. Design We have directly sequenced the DAX-1 gene from PCR-amplified DNA. The effect of the mutations on protein localization was assessed by immunocytochemistry in transfected cells. The impact of mutations on transcriptional activity was determined by transfection using a StAR promoter-luciferase reporter system. Results The mutation Y214X produces a protein lacking the C-terminal half of DAX-1. The other one, I361T, affects a highly conserved amino acid within the putative ligand-binding domain. The mutant Y214X displayed a wild-type subcellular localization while I361T was partially retained in the cytoplasm. Both mutants retained subtantial transcriptional repressive activity, compared to mutants producing early onset adrenal failure. Interestingly, I361T displayed similar in vitro transcriptional repressive activity to the mutant I439S previously described in a case of late-onset AHC. This shows that molecular alterations of DAX-1 leading to similar in vitro activities may result in very different ages of onset of adrenal failure, which suggests that additional genetic and epigenetic factors are important in determining the clinical course of AHC. Conclusions Our results help the understanding of structure–function relationships in the DAX-1 molecule, suggesting that the N-terminal domain is relatively autonomous and add credence to presumed molecular interactions within ligand binding domain of the protein. [ABSTRACT FROM AUTHOR]

In the course of positional cloning of the Congenital Dyserythropoietic Anemia type I (CDAI) [MIM 224120] gene on 15q15.1-15.3, we examined a family of French origin, in which the propositus suffered from asthenoteratozoospermia and nonsyndromic deafness in addition to CDAI. Two of his brothers had a similar phenotype. All three siblings were homozygous carriers of the CDA1 mutation as well as of a distally located ∼70 kb deletion of the proximal copy of a 106 kb tandem repeat on chromosome 15q15. These repeats encode four genes whose distal copies may be considered pseudogenes. Lack of functional stereocilin and CATSPER2 (a voltage-gate cation channel expressed specifically in spermatozoa) may explain the observed deafness and male infertility phenotypes. To the best of our knowledge, the involvement of CATSPER2 in asthenoteratozoospermia is the first description of a human autosomal gene defect associated with nonsyndromic male infertility. [ABSTRACT FROM AUTHOR]

We previously demonstrated that the gene tyke rescues the phenotype of a human mutant cell line unresponsive to α (IFN) and partially responsive to IFN-β. Here, we describe functional complementation of the mutant cells with the corresponding cDNA. To characterize the putative non-receptor protein tyrosine kinase encoded by the gene tyke and begin to understand its functioning, we have raised polyclonal antibodies against a segment of the protein. Using these, we have identified tyke as a 134-kDa protein which is rapidly and transiently phosphorylated on tyrosine in response to IFN-α/β and possesses an inducible kinase activity when tested in vitro. IFN-γ has no effect on the phosphorylation state of the protein. In agreement with previous genetic evidence, these results assign a role to tyke in the IFN-α/β signaling pathway and not in the IFN-γ pathway. Fractionation of cell lysates have helped to localize the bulk of the protein in the cytoplasm, with a minor fraction associated with the cell membrane. Both protein pools undergo activation upon short-term IFN treatment of intact cells. Through the study of the effect of pervanadate on the phosphorylation level and the activity of tyke, we conclude that activation of tyke by IFN-α does not require an intermediate regulatory tyrosine phosphatase. [ABSTRACT FROM AUTHOR]

Describes the genetic control mechanism which have involved in the determination of sex in fruit fly and nematode. Identification and isolation of mutations; Factors responsible for initiating testes formation; Measurement of the levels of X-linked transcripts; Inactivation of one of the two chromosomes during female development.

The article focuses on the premature ovarian failure (POF) in women. POF leads to female infertility. This disease is characterized by high plasma gonadotrophins levels and amenorrhoea (among persons under 40 years). BMP15 mutation in a POF case led to genetic screening of a panel of women revealing further potentially pathogenic variants. The finding of the study and the effects of mutations are highlighted in the article.

Abstract: Sox9 is a transcription factor that is critical for chondrogenesis, testis determination, and development of several other organs in vertebrates. Thus the levels of Sox9 protein and its activity may be tightly regulated. Here we show that inhibitors of the 26S proteasome increase both the levels of Sox9 protein and its transcriptional activity measured with Col2a1 promoter/enhancer construct in RCS cells and C3H10T1/2 cells. Indeed, in intact cells ubiquitination assays indicate that Sox9 is multiply ubiquitinated. The K398A mutation, which was introduced in a potential ubiquitin-binding site, increases the stability of Sox9 protein and its transcriptional activity of Col2a1, Col11a2, and AMH promoter/enhancer constructs without affecting the subcellular localization and the DNA binding efficiency of Sox9. Pulse-chase experiments show that the increased Sox9 levels resulting from treatment with the MG132 proteasome inhibitor or from the K398A mutation produce stabilization of the protein. Our in vitro studies indicate that the ubiquitin–proteasome proteolytic system degrades Sox9 and regulates its transcriptional activity. [Copyright &y& Elsevier]

WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis. Background. Donor splice-site de novo heterozygous mutations in intron 9 of the Wilms' tumor gene (WT1 ) have been reported in Frasier syndrome, which is defined by the association of focal and segmental glomerulosclerosis (FSGS), male pseudohermaphroditism, and gonadoblastoma. These splice-site mutations alter the WT1 alternative splicing leading to two WT1 isoforms, with (+) or without (-) three amino acids, lysine-threonine-serine (KTS), between zinc fingers 3 and 4. The aim of this work was to investigate the possibility that some cases of primary steroid-resistant nephrotic syndrome associated with FSGS may be caused by WT1 splice-site mutations. Methods. We analyzed WT1 exons 8 and 9 and the surrounding exon/intron boundary DNA sequences in 37 children with nonfamilial primary steroid-resistant nephrotic syndrome. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the relative ratio of +KTS/-KTS transcripts from immortalized lymphocyte RNA. Results. One boy with FSGS and associated pathologies (diaphragmatic hernia, proximal hypospadias, and unilateral testicular ectopia) was found to carry the heterozygous 1228 +4 C→T splice-site mutation. RT-PCR quantitation of the +KTS/-KTS transcripts from immortalized lymphocyte RNA of this patient showed a diminution of the +KTS/-KTS isoform ratio (0.43), which is identical to that reported in patients with Frasier syndrome. Using the same approach, healthy control subjects have +KTS/-KTS ratios ranging from 1.50 to 2.00. Conclusions. This study expands the range of the phenotypic presentation of the intron 9 splice-site WT1 mutations and adds to the already reported heterogeneity of primary steroid-resistant nephrotic syndromes. We suggest that these mutations are not likely to be a common cause of isolated steroid-resistant nephrotic syndrome, and... [ABSTRACT FROM AUTHOR]