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.

High-resolution array-CGH analysis on 46,XX patients affected by early onset primary ovarian insufficiency discloses new genes involved in ovarian function.

STUDY QUESTION: Can high resolution array-CGH analysis on a cohort of women showing a primary ovarian insufficiency (POI) phenotype in young age identify copy number variants (CNVs) with a deleterious effect on ovarian function? SUMMARY ANSWER: This approach has proved effective to clarify the role of CNVs in POI pathogenesis and to better unveil both novel candidate genes and pathogenic mechanisms. WHAT IS KNOWN ALREADY: POI describes the progression toward the cessation of ovarian function before the age of 40 years. Genetic causes are highly heterogeneous and despite several genes being associated with ovarian failure, most of genetic basis of POI still needs to be elucidated. STUDY DESIGN, SIZE, DURATION: The current study included 67 46,XX patients with early onset POI (<19 years) and 134 control females recruited between 2012 and 2016 at the Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano. PARTICIPANTS/MATERIALS, SETTING, METHODS: High resolution array-CGH analysis was carried out on POI patients' DNA. Results of patients and female controls were analyzed to search for rare CNVs. All variants were validated and subjected to a gene content analysis and disease gene prioritization based on the present literature to find out new ovary candidate genes. Case-control study with statistical analysis was carried out to validate our approach and evaluate any ovary CNVs/gene enrichment. Characterization of particular CNVs with molecular and functional studies was performed to assess their pathogenic involvement in POI. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 37 ovary-related CNVs involving 44 genes with a role in ovary in 32 patients. All except one of the selected CNVs were not observed in the control group. Possible involvement of the CNVs in POI pathogenesis was further corroborated by a case-control analysis that showed a significant enrichment of ovary-related CNVs/genes in patients (P = 0.0132; P = 0.0126). Disease gene prioritization identified both previously reported POI genes (e.g. BMP15, DIAPH2, CPEB1, BNC1) and new candidates supported by transcript and functional studies, such as TP63 with a role in oocyte genomic integrity and VLDLR which is involved in steroidogenesis. LARGE SCALE DATA: ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/); accession numbers SCV000787656 to SCV000787743. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis for almost all of the CNVs identified. Inheritance studies of CNVs in some non-familial sporadic cases was not performed as the parents' DNA samples were not available. Addionally, RT-qPCR analyses were carried out in few cases as RNA samples were not always available and the genes were not expressed in blood. WIDER IMPLICATIONS OF THE FINDINGS: Our array-CGH screening turned out to be efficient in identifying different CNVs possibly implicated in disease onset, thus supporting the extremely wide genetic heterogeneity of POI. Since almost 50% of cases are negative rare ovary-related CNVs, array-CGH together with next generation sequencing might represent the most suitable approach to obtain a comprehensive genetic characterization of POI patients. STUDY FUNDING/COMPETING INTEREST(S): Supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C203_2012) and 'Ricerca Finalizzata' (GR-2011-02351636, BIOEFFECT) to IRCCS Istituto Auxologico Italiano.

A balanced reciprocal translocation t(10;15)(q22.3;q26.1) interrupting ACAN gene in a family with proportionate short stature.

PURPOSE: Few examples of the involvement of a single gene in idiopathic short stature have been described until now. Our aim was to identify the causative gene of proportionate short stature in a large family showing co-segregation of the phenotype with the reciprocal translocation t(10;15)(q22;q24). METHODS: FISH mapping was carried out with BACs and long-range PCR probes to identify the smallest genomic regions harboring the translocation breakpoints. Real-Time RT-PCR was performed in blood after pre-amplification of target genes cDNA. RESULT: The affected family members presented with a final height of between - 2.41 and - 4.18 SDS and very mild skeletal dysmorphisms. Growth rates of the proband and of her cousin, whose childhood and pre-pubertal bone age corresponded to the chronological age, showed a poor growth spurt during treatment with rhGH. However, their adult height was greater than that of their untreated mothers, suggesting efficacy of GH therapy. Breakpoint mapping revealed that the translocation t(10;15)(q22.3;q26.1) disrupts, on 15q, the ACAN gene at intron 1, decreasing its transcriptional expression. CONCLUSIONS: This is the first description of a chromosome rearrangement disrupting ACAN and leading to its haploinsufficiency. ACAN loss of function should be considered a potential underpinning of short patients who display a poor growth spurt and belong to families with autosomal dominant segregation of proportionate short stature. Besides this core phenotype, literature review suggests that advanced bone age, early onset osteochondritis dissecans, osteoarthritis, intervertebral disc disease as well as craniofacial dysmorphisms can be important suggestive phenotypes in affected families.