Targeted whole exome sequencing and Drosophila modelling to unveil the molecular basis of primary ovarian insufficiency.

STUDY QUESTION: Can a targeted whole exome sequencing (WES) on a cohort of women showing a primary ovarian insufficiency (POI) phenotype at a young age, combined with a study of copy number variations, identify variants in candidate genes confirming their deleterious effect on ovarian function? SUMMARY ANSWER: This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis. WHAT IS KNOWN ALREADY: POI, a condition occurring in 1% of women under 40 years of age, affects women's fertility leading to a premature loss of ovarian reserve. The genetic causes of POI are highly heterogeneous and several determinants contributing to its prominent oligogenic inheritance pattern still need to be elucidated. STUDY DESIGN, SIZE, DURATION: WES screening for pathogenic variants of 41 Italian women with non-syndromic primary and early secondary amenorrhoea occurring before age 25 was replicated on another 60 POI patients, including 35 French and 25 American women, to reveal statistically significant shared variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: The Italian POI patients' DNA were processed by targeted WES including 542 RefSeq genes expressed or functioning during distinct reproductive or ovarian processes (e.g. DNA repair, meiosis, oocyte maturation, folliculogenesis and menopause). Extremely rare variants were filtered and selected by means of a Fisher Exact test using several publicly available datasets. A case-control Burden test was applied to highlight the most significant genes using two ad-hoc control female cohorts. To support the obtained data, the identified genes were screened on a novel cohort of 60 Caucasian POI patients and the same case-control analysis was carried out. Comparative analysis of the human identified genes was performed on mouse and Drosophila melanogaster by analysing the orthologous genes in their ovarian phenotype, and two of the selected genes were fruit fly modelled to explore their role in fertility. MAIN RESULTS AND THE ROLE OF CHANCE: The filtering steps applied to search for extremely rare pathogenic variants in the Italian cohort revealed 64 validated single-nucleotide variants/Indels in 59 genes in 30 out of 41 screened women. Burden test analysis highlighted 13 ovarian genes as being the most enriched and significant. To validate these findings, filtering steps and Burden analysis on the second cohort of Caucasian patients yielded 11 significantly enriched genes. Among them, AFP, DMRT3, MOV10, FYN and MYC were significant in both patient cohorts and hence were considered strong candidates for POI. Mouse and Drosophila comparative analysis evaluated a conserved role through the evolution of several candidates, and functional studies using a Drosophila model, when applicable, supported the conserved role of the MOV10 armitage and DMRT3 dmrt93B orthologues in female fertility. LARGE SCALE DATA: The datasets for the Italian cohort generated during the current study are publicly available at ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/): accession numbers SCV001364312 to SCV001364375. LIMITATIONS, REASONS FOR CAUTION: This is a targeted WES analysis hunting variants in candidate genes previously identified by different genomic approaches. For most of the investigated sporadic cases, we could not track the parental inheritance, due to unavailability of the parents' DNA samples; in addition, we might have overlooked additional rare variants in novel candidate POI genes extracted from the exome data. On the contrary, we might have considered some inherited variants whose clinical significance is uncertain and might not be causative for the patients' phenotype. Additionally, as regards the Drosophila model, it will be extremely important in the future to have more mutants or RNAi strains available for each candidate gene in order to validate their role in POI pathogenesis. WIDER IMPLICATIONS OF THE FINDINGS: The genomic, statistical, comparative and functional approaches integrated in our study convincingly support the extremely heterogeneous oligogenic nature of POI, and confirm the maintenance across the evolution of some key genes safeguarding fertility and successful reproduction. Two principal classes of genes were identified: (i) genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and (ii) genes safeguarding cell maintenance (piRNA and DNA repair pathways). STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C621_2016 and 08C924_2019) provided to IRCCS Istituto Auxologico Italiano, and by 'Piano Sostegno alla Ricerca' (PSR2020_FINELLI_LINEA_B) provided by the University of Milan; M.P.B. was supported by Telethon-Italy (grant number GG14181). There are no conflicts of interest.

Gene expression analysis reveals an angiogenic profile in uterine leiomyoma pseudocapsule.

The pseudocapsule (PC) of the uterine leiomyoma (UL) is an anatomic entity that surrounds the myoma separating it from the myometrium (UM). Although a number of microarray experiments have identified differences in gene expression profile in the UL when compared with the UM, there is a lack of systematic studies on the PC. In this study, quantitative RT-PCR analysis was performed on 18 matched PC, UL and UM specimens and results showed that the PC displays a specific gene expression profile. The low expression level of insulin-like growth factor (IGF-2), a fibroid specific marker, that we found in the PC and the UM when compared with the UL, clearly indicates that the PC is in structural continuity with the UM. However, the significant increase in endoglin expression level in PC with respect to the UL and UM indicates that an active neoangiogenesis is present in PC. Conversely, other angiogenic factors such as von Willebrand factor (vWF) and vascular endothelial growth factor A (VEGF-A) seem to have little influence on the PC angiogenesis. Because the endoglin is preferentially expressed in proliferating endothelial cells, whereas the vWF and VEGF-A are preferentially expressed in preexisting endothelial cells, our idea is that the angiogenic activity in the PC is linked to wound healing. The angiogenic activity is also sustained by intermediate expression level of cystein-rich angiogenesis inducer 61, connective tissue growth factor and collagen 4α2 genes all involved in the neoangiogenesis, that we detected in the PC. Taken together our data demonstrate that the specific expression pattern observed in the PC could be the response of the uterine wall's smooth cells to the tension imposed by the tumor. As a consequence, a neovascular structure is generated involving regenerative processes. For these reasons, we suggest that the laparoscopic intracapsular myomectomy (LIM), a new surgical technique that preserves the PC during the UL removal, should always be preferred, to favor a faster and proper uterine healing.

Occurrence of nonceliac gluten sensitivity in patients with allergic disease.

BACKGROUND: The aim of this study was to determine the occurrence of gluten sensitivity (GS) in a group of allergic patients and to assess the efficacy of a gluten-free diet (GFD) on the improvement of the symptomatology in those who were diagnosed with GS. METHODS: 262 unrelated allergic patients with gastrointestinal symptoms of obscure origin were tested for GS condition by biopsy. All patients were also genotyped for the typical celiac DQ2 and DQ8 molecules and investigated for several hematological parameters such as antigliadin and antiendomysial antibodies. Patients displaying mucosal lesions were invited to follow a GFD. RESULTS: Seventy-seven of the 262 allergic patients were positive to mucosal lesions, but negative to the antiAGA, antiEMA and to DQ2 and DQ8 molecules. We found, instead, a prevalence of the DQA1*05 allele, whereas anemia of inflammatory origin represented the predominant complaint in our subjects. The positive patients, who, after the GS diagnosis, followed a GFD, exhibited control of symptoms as well as stabilization of the hematological parameters even if allergic manifestations were not abated. CONCLUSIONS: A nonceliac gluten-sensitive enteropathy (NCGSE) commonly occurs in allergic patients. Based on the high prevalence of NCGSE in allergy, it is recommended that biopsy should be part of the routine investigation of allergic disease to offer the benefits of treatment with a GFD to the patients.

Homologous nuclear genes encode cytoplasmic and mitochondrial glutamine synthetase in Drosophila melanogaster.

We describe the cloning of the glutamine synthetase 1 (GS1) gene based on cross-homology with the glutamine synthetase 2 (GS2) gene in Drosophila melanogaster. We have determined the GS gene number in the Drosophila genome, and we describe the isolation of cDNA clones corresponding to the two isoforms, their entire sequence and their transcription pattern. We looked for subcellular localization of one enzymic isoform; in this way, we were able to locate the GS1 enzyme within the mitochondria of D. melanogaster. We have compared different GS sequences from plants and humans; emerging evolutionary implications are discussed. In addition, we have identified a certain highly stable secondary structure at the nucleotide level in the coding region of isoforms located in the organella.

Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster.

The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste+ flies and star-shaped in Ste flies, once again as observed in cry- males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry- males, a 0.7-kb mRNA is produced.

Drosophila melanogaster acylphosphatase: a common ancestor for acylphosphatase isoenzymes of vertebrate species.

An open reading frame encoding a putative acylphosphatase was found in Drosophila melanogaster. The corresponding gene product shows 40% identity and 22 additional amino acid residues at the C-terminus as compared to muscle- and common-type human acylphosphatases. Moreover, all the residues involved in the catalytic mechanism of vertebrate enzymes are conserved in the D. melanogaster acylphosphatase. The D. melanogaster protein and a deletion mutant, similar in length to vertebrate acylphosphatases, were produced by cloning the corresponding cDNA in Escherichia coli. The wild-type enzyme is a protein with a well-established three-dimensional fold and a markedly reduced conformational stability as compared to vertebrate isoenzymes. The specific activity of the enzyme is significantly lower than that found in vertebrate enzymes though the substrate binding capability is basically unaltered. The deletion of 22 residues does not cause a significant change in k(cat), while affecting the apparent binding parameters. This work suggests that the genes encoding the vertebrate enzymes originate from an ancestor gene by duplication and subsequent evolution.

Mutations in the glutamine synthetase I (gsI) gene produce embryo-lethal female sterility in Drosophila melanogaster.

A female-sterile mutation (fs(2) PM11-19) was recovered in a screen for P-M hybrid dysgenesis induced mutations uncovered by a deletion of region 21B and was identified as an allele of the gene encoding the Drosophila glutamine synthetase I (GSI) mitochondrial isozyme. Molecular analysis has shown that fs(2)PM11-19 contains a 5 kb insert within 500 bp upstream of the transcriptional start site of the gsI gene. Mutant flies have extremely low levels of gsI transcription and GSI activity. A pre-existing deficiency (Df(2L) netPM1) with a breakpoint near the transcription start site was also found to be a female-sterile allele of gsI. All eggs laid by PM11-19 homozygous females, as well as by females heterozygous for this mutation and a deletion or any of several recessive lethal alleles of the gsI gene, fail to hatch. We conclude that an adequate level of maternally supplied GSI activity is necessary in the early stages of Drosophila embryonic development.

Multiple upstream regulatory elements control the expression of the Drosophila white gene.

Constructions containing the Drosophila white gene and different amounts and arrangements of its regulatory region were introduced into the germ line of white mutant flies by P-mediated transformation. The results obtained with the different transposon constructions show that different parts of the 1.8-kb region preceding the transcription start are required for the expression of the gene in different tissues and at different developmental stages. Different sequences independently control the expression of the gene in the adult testes, in the larval and adult Malpighian tubules and in the eye. Another sequence located greater than 1080 bp upstream of the transcription start is the target of zeste interaction. The results also suggest that sequences required for dosage compensation are contained between -216 and the transcription start site. We show that at least some of these regulatory elements are equally functional if their distance from the promoter is varied or if their orientation is inverted. Their properties suggest that they act as enhancer-like elements to regulate the activity of the white promoter and, at least in the case of the zeste regulatory site, that they can act also in 'trans' on a white promoter locked in close physical proximity by homologous chromosome pairing.

Genetic determinants of glutamine synthetase in Drosophila melanogaster: a gene for glutamine synthetase I resides in the 21B3-6 region.

Recombinational and deletion mapping of electrophoretic variants of the glutamine synthetase I isozyme (GSI) in Drosophila melanogaster locates the gene in the 21B region on the second chromosome. We have conducted a genetic analysis of the region extending cytologically from 21A to 21B4-6. Recessive lethal mutations were generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU) mutagenesis and by hybrid dysgenesis (HD). These lethals fall into seven functional groups, which were partially ordered by complementation with cytologically defined deficiencies of this region generated by hybrid dysgenesis. Two of the EMS- and two of the ENU-induced lethals fulfill biochemical criteria expected for null alleles of the GSI gene.

Genetic, molecular and developmental analysis of the glutamine synthetase isozymes of Drosophila melanogaster.

The glutamine synthetase isozymes of Drosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. In Drosophila melanogaster two GS isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the two Drosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.

Glutamine synthetase gene evolution: a good molecular clock.

Glutamine synthetase (EC 6.3.1.2) gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. Our calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. Our data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves.

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

Interaction systems between heterochromatin and euchromatin in Drosophila melanogaster.

The constitutive heterochromatin is still one of the major unsolved problems in genetics. In Drosophila melanogaster three genetic systems involving specific interactions between heterochromatic and euchromatic genetic elements are known: the Segregation Distortion, the crystal-Stellate and the abo-ABO systems. The genetic and molecular analysis of each system will allow the identification of all the components and the elucidation of the mechanisms underlying their interactions. The results of this analysis should provide insights into the biological significance of heterochromatin and into the evolutionary forces that result in the maintainance and stability of this enigmatic genetic material.