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.

Ab Initio Computation of Charge Densities for Sn and Xe Isotopes.

We present the first ab initio calculations for open-shell nuclei past the tin isotopic line, focusing on Xe isotopes as well as doubly magic Sn isotopes. We show that, even for moderately hard interactions, it is possible to obtain meaningful predictions and that the NNLO_{sat} chiral interaction predicts radii and charge density distributions close to the experiment. We then make a new prediction for ^{100}Sn. This paves the way for ab initio studies of exotic charge density distributions at the limit of the present ab initio mass domain, where experimental data is becoming available. The present study closes the gap between the largest isotopes reachable by ab initio methods and the smallest exotic nuclei accessible to electron scattering experiments.

Developmental disorders with intellectual disability driven by chromatin dysregulation: Clinical overlaps and molecular mechanisms.

Advances in genomic analyses based on next-generation sequencing and integrated omics approaches, have accelerated in an unprecedented way the discovery of causative genes of developmental delay (DD) and intellectual disability (ID) disorders. Chromatin dysregulation has been recognized as common pathomechanism of mendelian DD/ID syndromes due to mutation in genes encoding chromatin regulators referred as transcriptomopathies or epigenetic disorders. Common to these syndromes are the wide phenotypic breadth and the recognition of groups of distinct syndromes with shared signs besides cognitive impairment, likely mirroring common molecular mechanisms. Disruption of chromatin-associated transcription machinery accounts for the phenotypic overlap of Cornelia de Lange with KBG and with syndromes of the epigenetic machinery. The genes responsible for Smith-Magenis-related disorders act in interconnected networks and the molecular signature of histone acetylation disorders joins Rubinstein-Taybi-related syndromes. Deciphering pathway interconnection of clinically similar ID syndromes may enhance search of common targets useful for developing new therapeutics.

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.

Clinical and molecular characterization of Rubinstein-Taybi syndrome patients carrying distinct novel mutations of the EP300 gene.

Rubinstein-Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by postnatal growth deficiency, skeletal abnormalities, dysmorphic features and cognitive deficit. Mutations in two genes, CREBBP and EP300, encoding two homologous transcriptional co-activators, have been identified in ˜55% and ˜3-5% of affected individuals, respectively. To date, only eight EP300-mutated RSTS patients have been described and 12 additional mutations are reported in the database LOVD. In this study, EP300 analysis was performed on 33 CREBBP-negative RSTS patients leading to the identification of six unreported germline EP300 alterations comprising one deletion and five point mutations. All six patients showed a convincing, albeit mild, RSTS phenotype with minor skeletal anomalies, slight cognitive impairment and few major malformations. Beyond the expansion of the RSTS-EP300-mutated cohort, this study indicates that EP300-related RSTS cases occur more frequently than previously thought (˜8% vs 3-5%); furthermore, the characterization of novel EP300 mutations in RSTS patients will enhance the clinical practice and genotype-phenotype correlations.

Molecular and genomic characterisation of cryptic chromosomal alterations leading to paternal duplication of the 11p15.5 Beckwith-Wiedemann region.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder with increased risk of paediatric tumours. The aetiology involves epigenetic and genetic alterations affecting the 11p15 region, methylation of the differentially methylated DMR2 region being the most common defect, while less frequent aetiologies include mosaic paternal 11p uniparental disomy (11patUPD), maternally inherited mutations of the CDKN1C gene, and hypermethylation of DMR1. A few patients have cytogenetic abnormalities involving 11p15.5. METHODS: Screening of 70 trios of BWS probands for 11p mosaic paternal UPD and for cryptic cytogenetic rearrangements using microsatellite segregation analysis identified a profile compatible with paternal 11p15 duplication in two patients. RESULTS: Fluorescence in situ hybridisation analysis revealed in one case the unbalanced translocation der(21)t(11;21)(p15.4;q22.3) originated from missegregation of a cryptic paternal balanced translocation. The second patient, trisomic for D11S1318, carried a small de novo dup(11)(p15.5p15.5), resulting from unequal recombination at paternal meiosis I. The duplicated region involves only IC1 and spares IC2/LIT1, as shown by fluorescent in situ hybridisation (FISH) mapping of the proximal duplication breakpoint within the amino-terminal part of KvLQT1. CONCLUSIONS: An additional patient with Wolf-Hirschorn syndrome was shown by FISH studies to carry a der(4)t(4;11)(p16.3;p15.4), contributed by a balanced translocation father. Interestingly, refined breakpoint mapping on 11p and the critical regions on the partner 21q and 4p chromosomal regions suggested that both translocations affecting 11p15.4 are mediated by segmental duplications. These findings of chromosomal rearrangements affecting 11p15.5-15.4 provide a tool to further dissect the genomics of the BWS region and the pathogenesis of this imprinting disorder.

Three-Territory DWI Acute Infarcts: Diagnostic Value in Cancer-Associated Hypercoagulation Stroke (Trousseau Syndrome).

BACKGROUND AND PURPOSE: DWI infarcts involving the bilateral anterior and posterior circulation suggest an embolic etiology. In the absence of an identifiable embolic source, we analyzed DWI lesions involving these 3 cerebral territories to determine the diagnostic value for ischemic infarction caused by cancer-associated hypercoagulation. MATERIALS AND METHODS: A retrospective analysis of all brain MR imaging studies at our institution from July 2014 to June 2015 was conducted, yielding 4075 studies. Of those, 17% (n = 709) contained the terms "restricted-diffusion" plus either "numerous," "innumerable," "multiple," or "bilateral." Of these 709 reports, 6% (n = 41) of DWI lesions involving 3 or more vascular territories of the bilateral anterior and posterior circulation were analyzed. RESULTS: Of the 41 patients, 19 separate etiologies were identified, the most frequent being malignancy-related infarctions (22% [n = 9]) and hypoxic-ischemic injury (12% [n = 5]). Only 2 patients had an indeterminate etiology. The most frequent etiology of infarctions not suspected clinically or radiographically was malignancy (P < .001). Infarctions of malignancy had a characteristic appearance, being nonenhancing, nonring-appearing clusters or single areas of restricted diffusion of 0.5-2 cm with a peripheral location or larger vascular territories, uncommonly in a watershed distribution, and with absence of diffuse cortical ribbon or deep gray nuclei involvement. CONCLUSIONS: Approximately 1 in 5 ischemic infarcts in patients with DWI lesions involving 3 vessel territories are malignancy related. In the absence of an identifiable embolic source, ischemic infarction with cancer-associated hypercoagulation accounts for 75% of cases. Cancer-associated hypercoagulation infarction should be considered, particularly when no other cause is apparent.

Cryptic subtelomeric translocation t(2;16)(q37;q24) segregating in a family with unexplained stillbirths and a dysmorphic, slightly retarded child.

We here describe a submicroscopic translocation affecting the subtelomeric regions of chromosomes 2q and 16q, and segregating in a family with stillbirths, early pregnancy losses, and two dysmorphic and slightly retarded babies. FISH analysis showed a 46,XY,der(2)t(2;16)(q37.3;q24.3) in the propositus, and a balanced t(2;16) in his mother, her conceptus and maternal grandfather. FISH with YACs and BACs made it possible to map the 2q37 breakpoint precisely between the regions covered by y952E1 and y746H1, and the 16q breakpoint between the regions encompassed by bA 309g16 and bA 533d19. The contribution of 2q37.3 monosomy and 16q24.3 trisomy to the proband's phenotype is compared with that in reported patients with similar imbalances of either chromosome.

Cerebral venous thrombosis associated with epoetin alfa therapy.

BACKGROUND: Cerebral venous thrombosis is a rare complication of polycythemia. To our knowledge, epoetin alfa-induced polycythemia has not previously been reported in association with cerebral venous thrombosis. CASE DESCRIPTION: A 37-year-old patient who was receiving peritoneal dialysis and epoetin alfa (Epogen) therapy presented with a several-day history of worsening headache, and a neuroimaging scan demonstrated thrombosis of the sagittal and transverse sinus. Epoetin alfa therapy, which had been initiated 3 months earlier according to an institutional protocol, was associated with a problematic increase in hematocrit values. CONCLUSIONS: Headache should raise the suspicion of cerebral venous thrombosis in patients who are being treated with epoetin alfa, particularly in the presence of elevated hematocrit values. Monitoring hematocrit parameters in accordance with "standard guidelines" is recommended.

Cerebral amyloid angiopathy manifesting as recurrent intracerebral hemorrhage.

Over a period of eight years, a normotensive woman experienced eight strokelike episodes. Computed tomographic (CT) scans obtained during each of the last seven episodes demonstrated intracerebral lobar hemorrhage. Cerebral angiography and contrast-enhanced CT scans demonstrated no underlying abnormality. Our patient had recurrent intracerebral hemorrhage (ICH) with no predisposing factors or dementia. The clinical diagnosis was primary cerebral amyloid angiopathy (CAA). Brain biopsy specimens demonstrated light microscopic and ultrastructural evidence of amyloid in cerebral arterioles. We believe that the combined clinical, CT, and ultrastructural changes in this case are unique. Recurrent ICH visualized by CT scanning has diagnostic value in CAA.

Ethylene oxide-induced polyneuropathy. A clinical and electrophysiologic study.

Occupational exposure to ethylene oxide (ETO) was manifested as a subacute polyneuropathy, with bilateral footdrop and denervation potentials on electromyography as the principal abnormalities in three young adults whom we examined. To our knowledge, this is the second report of neurotoxic effects caused by long-term ETO exposure in humans and brings the number of patients described with symptomatic polyneuropathy to five.

Coital cerebral hemorrhage.

Sudden headache, confusion, and hemiparesis followed within minutes of late-morning sexual intercourse in a 39-year-old man. Imaging studies revealed hemorrhage into a vascular malformation. Sexual intercourse and circadian rhythm independently elevate blood pressure, and their concurrence may predispose to cerebral hemorrhage in the presence of a vascular malformation.

Seizure as a cause of fracture.

Of 2,800 patients admitted to hospital with a diagnosis of seizure, 1.1% (30/2,800) sustained fracture. Of these, 0.5% (15/2,800) had fracture due to direct trauma, 0.3% (7/2,800) had fracture as a consequence of seizure alone, and in 0.3% (8/2,800) the etiology was not determined. In the trauma group, 11 of 17 fractures involved the skull, nasal bones, and clavicle, while in the nontrauma group, the proximal humerus was the site in 6 of 9 fractures. These findings indicate that fracture is an uncommon complication of seizure and is extremely rare in the absence of trauma where, however, the fracture may be pathognomonic (bilateral posterior dislocation or fracture-dislocation of the shoulder) or highly suggestive (unilateral posterior dislocation, fracture-dislocation of the shoulder) of seizure.

Myasthenic abduction mystagmus in a patient with hyperthyroidism.

Intravenous edrophonium chloride promptly stopped a sustained high frequency abduction nystagmus, supporting a diagnosis of myasthenia gravis in a patient with hyperthyroidism. Myasthenia gravis should be considered in the differential diagnosis of any acquired, isolated, and sustained abduction nystagmus, and this possibility should be ruled out by intravenous edrophonium chloride.

Adult celiac disease presenting as cerebellar syndrome.

A progressive pancerebellar syndrome in a 57-year-old man heralded what was subsequently diagnosed by malabsorption studies and jejunal biopsy as adult celiac disease. Postmortem examination demonstrated characteristic gastrointestinal and cerebral abnormalities associated with this enteropathy. The neuropathology underlying the ataxia, as well as the clinical features of palatal myoclonus and marked speech impairment, included marked cerebellar cortical atrophy with cell loss in dentate and olivary nuclei. Intestinal-absorption studies are indicated to evaluate patients with any neurologic illness that may be related to malabsorption.

Idiopathic granulomatous angiitis of the CNS manifesting as diffuse white matter disease.

A 48-year-old man presented with progressive spastic paraparesis and diffuse white matter involvement on neuroimaging that suggested a primary demyelinating disease. Brain biopsy 3 years after onset of symptoms demonstrated idiopathic granulomatous angiitis. In patients with MRI features of diffuse white matter disease of uncertain etiology, open brain biopsy of leptomeninges and parenchyma should be considered to exclude idiopathic granulomatous angiitis of the CNS.

FISH characterization of a supernumerary r(1)(::cen-->q22::q22-->sq21::) chromosome associated with multiple anomalies and bilateral cataracts.

We describe the case of a 15-year-old girl with multiple congenital anomalies, dysmorphic features, severe kyphoscoliosis, growth and mental retardation, and the absence of speech, in whom 35% of the cells carried a supernumerary ring chromosome 1. Fluorescence in situ hybridization (FISH) analysis using YAC/BAC clones spanning the region from 1p13 to 1q21 made it possible to determine the genomic content and structure of the ring(1), which was found to consist of the cytogenetic bands 1q21-22. A complex structure was delineated in the ring chromosome with a partial inverted duplication delimited by markers WI-7732 and WI-607, with WI-7396 and WI-8386 being the boundaries of the single copy segment. Comparison of the clinical signs of other patients with mosaic r(1) reported in the literature allowed the identification of a patient sharing a number of clinical signs including cataracts. Given that mutations of the GJA8 gene encoding connexin 50 (Cx50) and mapping to 1q21 have been associated with the presence of cataracts, it is possible that a gain in copy number or a rearrangement of GJA8 may contribute to cataractogenesis.

Refined FISH characterization of a de novo 1p22-p36.2 paracentric inversion and associated 1p21-22 deletion in a patient with signs of 1p36 microdeletion syndrome.

We report on a 10-year-old boy presenting with obesity, moderate mental retardation, large anterior fontanelle at birth, mild physical anomalies including mid-face hypoplasia, deep-set eyes, long philtrum, and small mouth. He was found to carry a paracentric inversion inv(1)(p22p36.2) associated with a 10 cM deletion at the proximal breakpoint. By YAC FISH, the boundaries of the deletion were established at IB1028 (1p21) and WI-5166 (1p22) STSs contained in YACs 781E8 and 954F6, respectively. This large region, covering about 10 cM, contains the COL11A1 and AMY2B genes, whose haploinsufficiency does not seem to contribute significantly to the clinical phenotype. On the other hand, the patient's clinical manifestations, also including visual problems and moderate mental retardation, are those typically observed in the 1p36 deletion syndrome. Refined mapping of the telomeric 1p36.2 inversion breakpoint was obtained by FISH of a PAC contig constructed to encompass this subinterval of the 1p36 microdeletion syndrome region. PACs 1024B10 and 884E7 were found to span the breakpoint, suggesting that the clinical signs of the 1p36 microdeletion syndrome might be due to disruption of a sequence lying at 1p36.2.

Pure 6p22-pter trisomic patient: refined FISH characterization and genotype-phenotype correlation.

First described in 1971, partial trisomy 6p is uncommon and generally secondary to a familial reciprocal translocation. The proximal breakpoint of the reported cases varies from p11 to p25. We here report on a patient with moderate mental retardation, craniofacial and pigmentary anomalies, proteinuria, and hyperglycemia who was found to have a mosaic karyotype 46,X,add(Y)(q12)/45,X. Fluorescence in situ hybridization (FISH) enabled us to identify that the additional material on Yqh derived from 6p and to define the rearrangement as der(Y)t(Y;6)(q12;p22). To the best of our knowledge, this is the first case of trisomy 6p22-pter without an associated deleted segment; the second breakpoint of the rearrangement is in Yqh. Precise mapping of the centromeric breakpoint of the trisomic 6p segment allowed a more convincing correlation between partial 6p trisomy and clinical phenotype to be addressed. In particular, the proteinuria often observed in 6p trisomic patients could be assigned to the 6p22-6pter region.